Methods of analysis of polymorphisms and uses thereof

ABSTRACT

The present invention provides methods for the assessment of diseases that result from the combined or interactive effects of two or more genetic variants, and in particular for diagnosing risk of developing such diseases in subjects using an analysis of genetic polymorphisms. Methods for the derivation of a net score indicative of a subject&#39;s risk of developing a disease are provided.

RELATED APPLICATIONS

This application claims priority to New Zealand Application Nos. 540249,filed May 20, 2005 and 541842, filed Aug. 15, 2005, both of which areincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is concerned with methods for the assessment ofdiseases that result from the combined or interactive effects of two ormore genetic variants, and in particular for diagnosing risk ofdeveloping such diseases in subjects using an analysis of geneticpolymorphisms.

BACKGROUND OF THE INVENTION

Diseases that result from the combined or interactive effects of two ormore genetic variants, with or without environmental factors, are calledcomplex diseases and include cancer, coronary artery disease, diabetes,stroke, and chronic obstructive pulmonary disease (COPD). Althoughcombining non-genetic risk factors to determine a risk level of outcomehas been in applied to coronary artery disease, (by combining individualfactors such as blood pressure, gender, fasting cholesterol, and smokingstatus), there are no such methods in combining the effects of multiplegenetic factors with non-genetic factors. There is a growing realizationthat the complex diseases, for which examples are given above, mayresult from the combined effects of common genetic variants orpolymorphisms rather than mutations which are rare (believed to bepresent in less than 1% of the general population). Moreover, theserelatively common polymorphisms can confer either susceptibility and/orprotective effects on the development of these diseases. In addition,the likelihood that these polymorphisms are actually expressed (termedpenetrance) as a disease or clinical manifestation requires a quantum ofenvironmental exposure before such a genetic tendency can be clinicallydetected.

SUMMARY OF THE INVENTION

Recent studies have identified a number of genetic variants orpolymorphisms that confer susceptibility to protection from COPD,occupational COPD (OCOPD), and lung cancer. The biological basis of justhow these polymorphisms interact or combine to determine risk remainsunclear.

Surprisingly, it has now been found that an assessment approach whichdetermines a subject's net score following the balancing of the numberof polymorphisms associated with protection from a disease against thenumber of polymorphisms associated with susceptibility to that diseasepresent in the subject is indicative of that subject's risk quotient.Furthermore, it has presently been determined that this approach iswidely applicable, on a disease-by-disease basis.

It is broadly to this approach to risk assessment that the presentinvention is directed.

Accordingly, in a first aspect, the present invention provides a methodof assessing a subject's risk of developing a disease which includes:

-   -   analysing a biological sample from said subject for the presence        or absence of protective polymorphisms and for the presence or        absence of susceptibility polymorphisms, wherein said protective        and susceptibility polymorphisms are associated with said        disease;    -   assigning a positive score for each protective polymorphism and        a negative score for each susceptibility polymorphism or vice        versa;    -   calculating a net score for said subject, said net score        representing the balance between the combined value of the        protective polymorphisms and the combined value of the        susceptibility polymorphisms present in the subject sample;    -   wherein a net protective score is predictive of a reduced risk        of developing said disease and a net susceptibility score is        predictive of an increased risk of developing said disease.

The value assigned to each protective polymorphism can be the same orcan be different. The value assigned to each susceptibility polymorphismcan be the same or can be different, with either each protectivepolymorphism having a negative value and each susceptibilitypolymorphism having a positive value, or vice versa. When the disease isa lung disease, the protective polymorphisms analysed can be selectedfrom one or more of the group consisting of: +760GG or +760CG within thegene encoding superoxide dismutase 3 (SOD3); −1296TT within the promoterof the gene encoding tissue inhibitor of metalloproteinase 3 (TIMP3); CC(homozygous P allele) within codon 10 of the gene encoding transforminggrowth factor beta (TGFβ); 2G2G within the promoter of the gene encodingmetalloproteinase 1 (MMP1); or one or more polymorphisms in linkagedisequilibrium with one or more of these polymorphisms.

Linkage disequilibrium is a phenomenon in genetics whereby two or moremutations or polymorphisms are in such close genetic proximity that theyare co-inherited. This means that in genotyping, detection of onepolymorphism as present implies the presence of the other. (Reich, D. E.et al. Linkage disequilibrium in the human genome. Nature 411:199-204(2001), herein incorporated by reference in its entirety).

Preferably, all polymorphisms of the group are analysed.

Preferably, the susceptibility polymorphisms analysed are selected fromone or more of the group consisting of: −82AA within the promoter of thegene encoding human macrophage elastase (MMP12); −1562CT or −1562TTwithin the promoter of the gene encoding metalloproteinase 9 (MMP9);1237AG or 1237AA (Tt or tt allele genotypes) within the 3′ region of thegene encoding a1-antitrypsin (a1AT); or one or more polymorphisms inlinkage disequilibrium with one or more of these polymorphisms.

Preferably, all polymorphisms of the group are analysed.

In one embodiment each protective polymorphism is assigned a value of −1and each susceptibility polymorphism is assigned a value of +1.

In another embodiment each protective polymorphism is assigned a valueof +1 and each susceptibility polymorphism is assigned a value of −1.

When the disease is COPD, the protective polymorphisms analysed can beselected from one or more of the group consisting of: −765 CC or CG inthe promoter of the gene encoding cyclooxygenase 2 (COX2); Arg 130 GlnAA in the gene encoding Interleukin-13 (IL-13); Asp 298 Glu TT in thegene encoding nitric oxide synthase 3 (NOS3); Lys 420 Thr AA or AC inthe gene encoding vitamin binding protein (VDBP); Glu 416 Asp TT or TGin the gene encoding VDBP; Ile 105 Val AA in the gene encodingglutathione S-transferase (GSTP1); MS in the gene encoding a1-antitrypsin (a 1 AT); the +489 GG geneotype in the gene encodingTissue Necrosis factor a (TNFa); the −308 GG geneotype in the geneencoding TNFa; the C89Y AA or AG geneotype in the gene encodoing SMAD3;the 161 GG genotype in the gene encodoing Mannose binding lectin 2(MBL2); the −1903 AA genotype in the gene encoding Chymase 1 (CMA1); theArg 197 Gln AA genotype in the gene encoding N-Acetyl transferase 2(NAT2); the His 139 Arg GG genotype in the gene encoding Microsomalepoxide hydrolase (MEH); the −366 AA or AG genotype in the gene encoding5 Lipo-oxygenase (ALOX5); the HOM T2437C TT genotype in the geneencoding Heat Shock Protein 70 (HSP 70); the exon 1 + 49 CT or TTgenotype in the gene encoding Elafin; the Gln 27 Glu GG genotype in thegene encoding 132 Adrenergic receptor (ADBR); the −1607 1G1G or 1G2Ggenotype in the promoter of the gene encoding Matrix Metalloproteinase 1(MMP1); or one or more polymorphisms in linkage disequilibrium with oneor more of these polymorphisms. Preferably, all polymorphisms of thegroup are analysed.

Preferably, the susceptibility polymorphisms analysed are selected fromone or more of the group consisting of: Arg 16 Gly GG in the geneencoding β2-adrenoreceptor (ADRB2); 105 AA in the gene encodingInterleukin-18 (IL-18); −133 CC in the promoter of the gene encodingIL-18; −675 5G5G in the promoter of the gene encoding plasminogenactivator inhibitor 1 (PAI-1); −1055 TT in the promoter of the geneencoding IL-13; 874 TT in the gene encoding interferon gamma (IFN?); the+489 AA or AG genotype in the gene encoding TNFa; the −308 AA or AGgenotype in the gene encoding TNFa; the C89Y GG genotype in the geneencoding SMAD3; the E469K GG genotype in the gene encoding IntracellularAdhesion molecule 1 (ICAM1); the Gly 881 Arg GC or CC genotype in thegene encoding Caspase (NOD2); the −511 GG genotype in the gene encodingIL1B; the Tyr 113 His TT genotype in the gene encoding MEH; the −366 GGgenotype in the gene encoding ALOX5; the HOM T2437C CC or CT genotype inthe gene encoding HSP 70; the +13924 AA genotype in the gene encodingChloride Channel Calcium-activated 1 (CLCA1); the −159 CC genotype inthe gene encoding Monocyte differentiation antigen CD-14 (CD-14); or oneor more polymorphisms in linkage disequilibrium with one or more ofthese polymorphisms.

Preferably, all polymorphisms of the group are analysed.

In one embodiment each protective polymorphism is assigned a value of −1and each susceptibility polymorphism is assigned a value of +1.

In one embodiment each protective polymorphism is assigned a value of +1and each susceptibility polymorphism is assigned a value of −1.

When the disease is OCOPD, the protective polymorphisms analysed can beselected from one or more of the group consisting of: −765 CC or CG inthe promoter of the gene encoding COX2; −251 AA In the promoter of thegene encoding interleukin-8 (IL-8); Lys 420 Thr AA in the gene encodingVDBP; Glu 416 Asp TT or TG in the gene encoding VDBP; exon 3 T/C RR inthe gene encoding microsomal epoxide hydrolase (MEH); Arg 312 Gln AG orGG in the gene encoding SOD3; MS or SS in the gene encoding a1AT; Asp299 Gly AG or GG in the gene encoding toll-like receptor 4 (TLR4); Gln27 Glu CC in the gene encoding ADRB2; −518 AA in the gene encodingIL-11; Asp 298 Glu TT in the gene encoding NOS3; or one or morepolymorphisms in linkage disequilibrium with one or more of thesepolymorphisms.

Preferably, all polymorphisms of the group are analysed.

Preferably, the susceptibility polymorphisms analysed are selected fromone or more of the group consisting of: −765 GG in the promoter of thegene encoding COX2; 105 AA in the gene encoding IL-18; −133 CC in thepromoter of the gene encoding IL-18; −675 5G5G in the promoter of thegene encoding PAI-1; Lys 420 Thr CC in the gene encoding VDBP; Glu 416Asp GG in the gene encoding VDBP; Ile 105 Val GG in the gene encodingGSTP1; Arg 312 Gln AA in the gene encoding SOD3; −1055 TT in thepromoter of the gene encoding IL-13; 3′ 1237 Tt or tt in the geneencoding a 1AT; −1607 2G2G in the promoter of the gene encoding MMP1; orone or more polymorphisms in linkage disequilibrium with one or more ofthese polymorphisms.

Preferably, all polymorphisms of the group are analysed.

In one embodiment each protective polymorphism is assigned a value of −1and each susceptibility polymorphism is assigned a value of +1.

In one embodiment each protective polymorphism is assigned a value of +1and each susceptibility polymorphism is assigned a value of −1.

When the disease is lung cancer, the protective polymorphisms analysedcan be selected from one or more of the group consisting of: the Asp 298Glu TT genotype in the gene encoding NOS3; the Arg 312 Gln CG or GGgenotype in the gene encoding SOD3; the Asn 357 Ser AG or GG genotype inthe gene encoding MMP12; the 105 AC or CC genotype in the gene encodingIL-18; the −133 CG or GG genotype in the gene encoding IL-18; the −765CC or CG genotype in the promoter of the gene encoding COX2; the −221 TTgenotype in the gene encoding Mucin 5AC (MUC5AC); the intron 1 C/T TTgenotype in the gene encoding Arginase 1 (Arg1); the Leu252Val GGgenotype in the gene encoding Insulin-like growth factor II receptor(IGF2R); the −1082 GG genotype in the gene encoding Interleukin 10(IL-10); the −251 AA genotype in the gene encoding Interleukin 8 (IL-8);the Arg 399 Gln AA genotype in the X-ray repair complementing defectivein Chinese hamster 1 (XRCC1) gene; the A870G GG genotype in the geneencoding cyclin D (CCND1); the −751 GG genotype in the promoter of thexeroderma pigmentosum complementation group D (XPD) gene; the Ile 462Val AG or GG genotype in the gene encoding cytochrome P450 1A1 (CYP1A1);the Ser 326 Cys GG genotype in the gene encoding 8-Oxoguanine DNAglycolase (OGG1); the Phe 257 Ser CC genotype in the gene encoding REV1;or one or more polymorphisms in linkage disequilibrium with any one ormore of these polymorphisms.

Preferably, all polymorphisms of the group are analysed.

Preferably, the susceptibility polymorphisms analysed are selected fromone or more of the group consisting of: the −786 TT genotype in thepromoter of the gene encoding NOS3; the Ala 15 Thr GG genotype in thegene encoding anti-chymotrypsin (ACT); the 105 AA genotype in the geneencoding IL-18; the −133 CC genotype in the promoter of the geneencoding IL-18; the 874 AA genotype in the gene encoding IFN?; the −765GG genotype in the promoter of the gene encoding COX2; the −447 CC or GCgenotype in the gene encoding Connective tissue growth factor (CTGF);and the +161 AA or AG genotype in the gene encoding MBL2; −511 GGgenotype in the gene encoding IL-1B; the A-670G AA genotype in the geneencoding FAS (Apo-1/CD95); the Arg 197 Gln GG genotype in the geneencoding N-acetyltransferase 2 (NAT2); the Ile462 Val AA genotype in thegene encoding CYP1A1; the 1019 G/C Pst I CC or CG genotype in the geneencoding cytochrome P450 2E1 (CYP2E1); the C/T Rsa I TT or TC genotypein the gene encoding CYP2E1; the GSTM null genotype in the gene encodingGSTM; the −1607 2G/2G genotype in the promoter of the gene encodingMMP1; the Gln 185 Glu CC genotype in the gene encoding Nibrin (NBS1);the Asp 148 Glu GG genotype in the gene encoding Apex nuclease (APE1);or one or more polymorphisms in linkage disequilibrium with any one ormore of these polymorphisms.

Preferably, all polymorphisms of the group are analysed.

In one embodiment each protective polymorphism is assigned a value of −1and each susceptibility polymorphism is assigned a value of +1.

In one embodiment each protective polymorphism is assigned a value of +1and each susceptibility polymorphism is assigned a value of −1.

In various embodiments the subject is or has been a smoker.

Preferably, the methods of the invention are performed in conjunctionwith an analysis of one or more risk factors, including one or moreepidemiological risk factors, associated with the risk of developing alung disease including COPD, emphysema, OCOPD, and lung cancer. Suchepidemiological risk factors include but are not limited to smoking orexposure to tobacco smoke, age, sex, and familial history.

In another aspect, the invention provides a method of determining asubject's risk of developing a disease, said method comprising

-   -   obtaining the result of one or more analyses of a sample from        said subject to determine the presence or absence of protective        polymorphisms and the presence or absence of susceptibility        polymorphisms, and wherein said protective and susceptibility        polymorphisms are associated with said disease;    -   assigning a positive score for each protective polymorphism and        a negative score for each susceptibility polymorphism or vice        versa;    -   calculating a net score for said subject, said net score        representing the balance between the combined value of the        protective polymorphisms and the combined value of the        susceptibility polymorphisms present in the subject sample;    -   wherein a net protective score is predictive of a reduced risk        of developing said disease and a net susceptibility score is        predictive of an increased risk of developing said disease.

In a further aspect the present invention provides a method forassessing the risk of a subject developing a disease which includes

-   -   determining a net score for said subject in accordance with the        methods of the invention described above, in combination with a        score based on the presence or absence of one or more        epidemiological risk factors,    -   wherein a net protective score is predictive of a reduced risk        of developing said disease and a net susceptibility score is        predictive of an increased predisposition and/or susceptibility        to said disease.

In another aspect, the present invention provides a kit for assessing asubject's risk of developing a disease, said kit comprising a means ofanalysing a sample from said subject for the presence or absence of oneor more protective polymorphisms and one or more susceptibilitypolymorphisms as described herein.

In yet a further aspect, the present invention provides a method ofprophylactic or therapeutic intervention in relation to a subject havinga net susceptibility score for a disease as determined by a method asdefined above which includes the steps of communicating to said subjectsaid net susceptibility score, and advising on changes to the subject'slifestyle that could reduce the risk of developing said disease.

In still a further aspect, the present invention provides a method oftreatment of a subject to decrease to the risk of developing a diseasethrough alteration of the net score for said subject as determined by amethod as defined above, wherein said method of treatment includesreversing, genotypically or phenotypically, the presence and/orfunctional effect of one or more susceptibility polymorphisms associatedwith said disease; and/or replicating and/or mimicking, genotypically orphenotypically, the presence and/or functional effect of one or moreprotective polymorphisms associated with said disease.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: depicts a graph showing combined frequencies of the presence orabsence of selected protective genotypes in the COPD subjects and inresistant smokers.

FIG. 2: depicts a graph showing net scores for protective andsusceptibility polymorphisms in COPD subjects.

FIG. 3: depicts a graph showing net scores for protective andsusceptibility polymorphisms in OCOPD subjects.

FIG. 4: depicts a graph showing net scores for protective andsusceptibility polymorphisms in subjects with lung cancer.

FIG. 5: depicts a graph showing net scores for protective andsusceptibility polymorphisms in subjects with lung cancer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

There is a need for a method for assessing a subject's risk ofdeveloping a disease using genetic (and optionally non-genetic) riskfactors. In some embodiments, it is an object of the present inventionto go some way towards meeting this need and/or to provide the publicwith a useful choice.

The present invention is directed to methods for the assessment of thegenetic risk quotient of a particular subject with respect to aparticular disease. The methods rely upon the recognition that for many(if not all) diseases there exist genetic polymorphisms which fall intotwo categories—namely those indicative of a reduced risk of developing aparticular disease (which can be termed “protective polymorphisms” or“protective SNPs”) and those indicative of an increased risk ofdeveloping a particular disease (which can be termed “susceptibilitypolymorphisms” or “susceptibility SNPs”).

As used herein, the phrase “risk of developing [a] disease” means thelikelihood that a subject to whom the risk applies will develop thedisease, and includes predisposition to, and potential onset of thedisease. Accordingly, the phrase “increased risk of developing [a]disease” means that a subject having such an increased risk possesses anhereditary inclination or tendency to develop the disease. This does notmean that such a person will actually develop the disease at any time,merely that he or she has a greater likelihood of developing the diseasecompared to the general population of individuals that either does notpossess a polymorphism associated with increased disease risk, or doespossess a polymorphism associated with decreased disease risk. Subjectswith an increased risk of developing the disease include those with apredisposition to the disease, for example in the case of COPD, atendency or prediliction regardless of their lung function at the timeof assessment, for example, a subject who is genetically inclined toCOPD but who has normal lung function, those at potential risk, forexample in the case of COPD, subjects with a tendency to mildly reducedlung function who are likely to go on to suffer COPD if they keepsmoking, and subjects with potential onset of the disease, for examplein the case of COPD, subjects who have a tendency to poor lung functionon spirometry etc., consistent with COPD at the time of assessment.

Similarly, the phrase “decreased risk of developing [a] disease” meansthat a subject having such a decreased risk possesses an hereditarydisinclination or reduced tendency to develop the disease. This does notmean that such a person will not develop the disease at any time, merelythat he or she has a decreased likelihood of developing the diseasecompared to the general population of individuals that either doespossess one or more polymorphisms associated with increased diseaserisk, or does not possess a polymorphism associated with decreaseddisease risk.

It will be understood that in the context of the present invention theterm “polymorphism” means the occurrence together in the same populationat a rate greater than that attributable to random mutation (usuallygreater than 1%) of two or more alternate forms (such as alleles orgenetic markers) of a chromosomal locus that differ in nucleotidesequence or have variable numbers of repeated nucleotide units. Seewww.ornl.gov/sci/techresources/Human_Genome/publicat/97pr/09gloss.html#p.Accordingly, the term “polymorphisms” is used herein contemplatesgenetic variations, including single nucleotide substitutions,insertions and deletions of nucleotides, repetitive sequences (such asmicrosatellites), and the total or partial absence of genes (eg. nullmutations). As used herein, the term “polymorphisms” also includesgenotypes and haplotypes. A genotype is the genetic composition at aspecific locus or set of loci. A haplotype is a set of closely linkedgenetic markers present on one chromosome which are not easily separableby recombination, tend to be inherited together, and can be in linkagedisequilibrium. A haplotype can be identified by patterns ofpolymorphisms such as SNPs. Similarly, the term “single nucleotidepolymorphism” or “SNP” in the context of the present invention includessingle base nucleotide substitutions and short deletion and insertionpolymorphisms. It will further be understood that the term “disease” isused herein in its widest possible sense, and includes conditions whichcan be considered disorders and/or illnesses which have a genetic basisor to which the genetic makeup of the subject contributes.

Using case-control studies, the frequencies of several genetic variants(polymorphisms) of candidate genes have been compared in diseasesufferers, for example, in chronic obstructive pulmonary disease (COPD)sufferers, in occupational chronic obstructive pulmonary disease (OCOPD)sufferers, and in lung cancer sufferers, and in control subjects notsuffering from the relevant disease, for example smokers without lungcancer and with normal lung function. The majority of these candidategenes have confirmed (or likely) functional effects on gene expressionor protein function.

In various specific embodiments, the frequencies of polymorphismsbetween blood donor controls, resistant subjects and those with COPD,the frequencies of polymorphisms between blood donor controls, resistantsubjects and those with OCOPD, and the frequencies of polymorphismsbetween blood donor controls, resistant subjects and those with lungcancer, have been compared. This has resulted in both protective andsusceptibility polymorphisms being identified for each disease.

The surprising finding relevant to this invention is that a combinedanalysis of protective and susceptibility polymorphisms discriminatoryfor a given disease yields a result that is indicative of that subject'srisk quotient for that disease. This approach is widely applicable, on adisease-by-disease basis.

The present invention identifies methods of assessing the risk of asubject developing a disease which includes determining in said subjectthe presence or absence of protective and susceptibility polymorphismsassociated with said disease. A net score for said subject is derived,said score representing the balance between the combined value of theprotective polymorphisms present in said subject and the combined valueof the susceptibility polymorphisms present in said subject. A netprotective score is predictive of a reduced risk of developing saiddisease, and a net susceptibility score is predictive of an increasedrisk of developing said disease.

Within each category (protective polymorphisms, susceptibilitypolymorphisms, respectively) the polymorphisms can each be assigned thesame value. For example, in the analyses presented in the Examplesherein, each protective polymorphism associated with a given disease isassigned a value of +1, and each susceptibility polymorphism is assigneda value of −1. Alternatively, polymorphisms discriminatory for a diseasewithin the same category can each be assigned a different value toreflect their discriminatory value for said disease. For example, apolymorphism highly discriminatory of risk of developing a disease canbe assigned a high weighting, for example a polymorphism with a highOdd's ratio can be considered highly discriminatory of disease, and canbe assigned a high weighting.

Accordingly, in a first aspect, the present invention provides a methodof assessing a subject's risk of developing a disease which includes:

analysing a biological sample from said subject for the presence orabsence of protective polymorphisms and for the presence or absence ofsusceptibility polymorphisms, wherein said protective and susceptibilitypolymorphisms are associated with said disease;

assigning a positive score for each protective polymorphism and anegative score for each susceptibility polymorphism or vice versa;

calculating a net score for said subject, said net score representingthe balance between the combined value of the protective polymorphismsand the combined value of the susceptibility polymorphisms present inthe subject sample;

wherein a net protective score is predictive of a reduced risk ofdeveloping said disease and a net susceptibility score is predictive ofan increased risk of developing said disease.

The subject sample can have already been analysed for the presence orabsence of one or more protective or susceptibility polymorphisms, andthe method includes the steps of

-   -   assigning a positive score for each protective polymorphism and        a negative score for each susceptibility polymorphism or vice        versa;    -   calculating a net score for said subject, said net score        representing the balance between the combined value of the        protective polymorphisms and the combined value of the        susceptibility polymorphisms present in the subject sample;    -   wherein a net protective score is predictive of a reduced risk        of developing said disease and a net susceptibility score is        predictive of an increased risk of developing said disease.

In one embodiment described herein in Example 1, 17 susceptibilitygenetic polymorphisms and 19 protective genetic polymorphisms identifiedas discriminatory for COPD were analysed using methods of the invention.These analyses can be used to determine the risk quotient of any subjectfor COPD, and in particular to identify subjects at greater risk ofdeveloping lung cancer.

In another embodiment described herein in Example 2, 11 susceptibilitygenetic polymorphisms and 11 protective genetic polymorphisms identifiedas discriminatory for OCOPD are analysed using methods of the invention.These analyses can be used to determine the risk quotient of any subjectfor OCOPD, and in particular to identify subjects at greater risk ofdeveloping OCOPD.

In a further embodiment described herein in Example 3, 19 susceptibilitygenetic polymorphisms and 17 protective genetic polymorphisms identifiedas discriminatory for lung cancer are analysed using methods of theinvention. These analyses can be used to determine the risk quotient ofany subject for lung cancer, and in particular to identify subjects atgreater risk of developing lung cancer.

Susceptibility and protective polymorphisms can readily be identifiedfor other diseases using approaches similar to those described in theExamples, as well as in PCT International Application No. PCT/NZ02/00106(published as WO 02/099134 and herein incorporated by reference in itsentirety) via which four susceptibility and three protectivepolymorphisms discriminatory for lung disease were identified.

The one or more polymorphisms can be detected directly or by detectionof one or more polymorphisms which are in linkage disequilibrium withsaid one or more polymorphisms. As discussed above, linkagedisequilibrium is a phenomenon in genetics whereby two or more mutationsor polymorphisms are in such close genetic proximity that they areco-inherited. This means that in genotyping, detection of onepolymorphism as present implies the presence of the other. (Reich D E etal; Linkage disequilibrium in the human genome, Nature 2001,411:199-204.)

Examples of polymorphisms reported to be in linkage disequilibrium arepresented herein, and include the Interleukin-18 −133 C/G and 105 A/Cpolymorphisms, and the Vitamin D binding protein Glu 416 Asp and Lys 420Thr polymorphisms, as shown below.

LD rs Alleles between Phenotype Gene SNPs numbers in LD alleles in COPDInterleukin- IL18 −133 rs360721 C allele Strong LD CC 18 C/G susceptibleIL18 105 rs549908 A allele AA A/C susceptible Vitamin D VDBP rs4588 Aallele Strong LD AA/AC binding Lys 420 protective protein Thr VDBPrs7041 T allele TT/TG Glu 416 protective Asp

It will be apparent that polymorphsisms in linkage disequilibrium withone or more other polymorphism associated with increased or decreasedrisk of developing COPD, emphysema, or both COPD and emphysema will alsoprovide utility as biomarkers for risk of developing COPD, emphysema, orboth COPD and emphysema. The data presented herein shows that thefrequency for SNPs in linkage disequilibrium is very similar.Accordingly, these genetically linked SNPs can be utilized in combinedpolymorphism analyses to derive a level of risk comparable to thatcalculated from the original SNP.

It will therefore be apparent that one or more polymorphisms in linkagedisequilibrium with the polymorphisms specified herein can beidentified, for example, using public data bases. Examples of suchpolymorphisms reported to be in linkage disequilibrium with thepolymorphisms specified herein are presented herein in Table 21.

The methods of the invention are primarily reliant on geneticinformation such as that derived from methods suitable to the detectionand identification of single nucleotide polymorphisms (SNPs) associatedwith the specific disease for which a risk assessment is desired. Insome embodiments, a SNP is a single base change or point mutationresulting in genetic variation between individuals. SNPs occur in thehuman genome approximately once every 100 to 300 bases, and can occur incoding or non-coding regions. Due to the redundancy of the genetic code,a SNP in the coding region may or may not change the amino acid sequenceof a protein product. A SNP in a non-coding region can, for example,alter gene expression by, for example, modifying control regions such aspromoters, transcription factor binding sites, processing sites,ribosomal binding sites, and affect gene transcription, processing, andtranslation.

SNPs can facilitate large-scale association genetics studies, and therehas recently been great interest in SNP discovery and detection. SNPsshow great promise as markers for a number of phenotypic traits(including latent traits), such as for example, disease propensity andseverity, wellness propensity, and drug responsiveness including, forexample, susceptibility to adverse drug reactions. Knowledge of theassociation of a particular SNP with a phenotypic trait, coupled withthe knowledge of whether an individual has said particular SNP, canenable the targeting of diagnostic, preventative and therapeuticapplications to allow better disease management, to enhanceunderstanding of disease states and to ultimately facilitate thediscovery of more effective treatments, such as personalised treatmentregimens.

Indeed, a number of databases have been constructed of known SNPs, andfor some such SNPs, the biological effect associated with a SNP. Forexample, the NCBI SNP database “dbSNP” is incorporated into NCBI'sEntrez system and can be queried using the same approach as the otherEntrez databases such as PubMed and GenBank. This database has recordsfor over 1.5 million SNPs mapped onto the human genome sequence. EachdbSNP entry includes the sequence context of the polymorphism (i.e., thesurrounding sequence), the occurrence frequency of the polymorphism (bypopulation or individual), and the experimental method(s), protocols,and conditions used to assay the variation, and can include informationassociating a SNP with a particular phenotypic trait.

At least in part because of the potential impact on health and wellness,there has been and continues to be a great deal of effort to developmethods that reliably and rapidly identify SNPs. This is no trivialtask, at least in part because of the complexity of human genomic DNA,with a haploid genome of 3×10⁹ base pairs, and the associatedsensitivity and discriminatory requirements.

Genotyping approaches to detect SNPs well-known in the art include DNAsequencing, methods that require allele specific hybridization ofprimers or probes, allele specific incorporation of nucleotides toprimers bound close to or adjacent to the polymorphisms (often referredto as “single base extension”, or “minisequencing”), allele-specificligation (joining) of oligonucleotides (ligation chain reaction orligation padlock probes), allele-specific cleavage of oligonucleotidesor PCR products by restriction enzymes (restriction fragment lengthpolymorphisms analysis or RFLP) or chemical or other agents, resolutionof allele-dependent differences in electrophoretic or chromatographicmobilities, by structure specific enzymes including invasive structurespecific enzymes, or mass spectrometry. Analysis of amino acid variationis also possible where the SNP lies in a coding region and results in anamino acid change.

DNA sequencing allows the direct determination and identification ofSNPs. The benefits in specificity and accuracy are generally outweighedfor screening purposes by the difficulties inherent in whole genome, oreven targeted subgenome, sequencing.

Mini-sequencing involves allowing a primer to hybridize to the DNAsequence adjacent to the SNP site on the test sample underinvestigation. The primer is extended by one nucleotide using all fourdifferentially tagged fluorescent dideoxynucleotides (A,C,G, or T), anda DNA polymerase. Only one of the four nucleotides (homozygous case) ortwo of the four nucleotides (heterozygous case) is incorporated. Thebase that is incorporated is complementary to the nucleotide at the SNPposition.

A number of methods currently used for SNP detection involvesite-specific and/or allele-specific hybridisation (Matsuzaki, H. et al.Genome Res. 14:414-425 (2004); Matsuzaki, H. et al. Nat. Methods1:109-111 (2004); Sethi, A. A. et al. Clin. Chem. 50(2):443-446 (2004),each of the foregoing which is herein incorporated by reference in itsentirety). These methods are largely reliant on the discriminatorybinding of oligonucleotides to target sequences containing the SNP ofinterest. The techniques of Affymetrix (Santa Clara, Calif.) and NanogenInc. (San Diego, Calif.) are particularly well-known, and utilize thefact that DNA duplexes containing single base mismatches are much lessstable than duplexes that are perfectly base-paired. The presence of amatched duplex is detected by fluorescence.

The majority of methods to detect or identify SNPs by site-specifichybridisation require target amplification by methods such as PCR toincrease sensitivity and specificity (see, for example U.S. Pat. No.5,679,524, PCT publication WO 98/59066, PCT publication WO 95/12607,each of the foregoing which is herein incorporated by reference in itsentirety). US Application 20050059030 (incorporated herein in itsentirety) describes a method for detecting a single nucleotidepolymorphism in total human DNA without prior amplification orcomplexity reduction to selectively enrich for the target sequence, andwithout the aid of any enzymatic reaction. The method utilises asingle-step hybridization involving two hybridization events:hybridization of a first portion of the target sequence to a captureprobe, and hybridization of a second portion of said target sequence toa detection probe. Both hybridization events happen in the samereaction, and the order in which hybridisation occurs is not critical.

US Application 20050042608 (herein incorporated by reference in itsentirety) describes a modification of the method of electrochemicaldetection of nucleic acid hybridization of Thorp et al. (U.S. Pat. No.5,871,918, herein incorporated by reference in its entirety). Briefly,capture probes are designed, each of which has a different SNP base anda sequence of probe bases on each side of the SNP base. The probe basesare complementary to the corresponding target sequence adjacent to theSNP site. Each capture probe is immobilized on a different electrodehaving a non-conductive outer layer on a conductive working surface of asubstrate. The extent of hybridization between each capture probe andthe nucleic acid target is detected by detecting the oxidation-reductionreaction at each electrode, utilizing a transition metal complex. Thesedifferences in the oxidation rates at the different electrodes are usedto determine whether the selected nucleic acid target has a singlenucleotide polymorphism at the selected SNP site.

The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPE™technology can genotype very large numbers of SNPs simultaneously fromsmall or large pools of genomic material. This technology usesfluorescently labeled probes and compares the collected genomes of twopopulations, enabling detection and recovery of DNA fragments spanningSNPs that distinguish the two populations, without requiring prior SNPmapping or knowledge.

A number of other methods for detecting and identifying SNPs exist.These include the use of mass spectrometry, for example, to measureprobes that hybridize to the SNP (Ross, P. L. et al. Discrimination ofsingle-nucleotide polymorphisms in human DNA using peptide nucleic acidprobes detected by MALDI-TOF mass spectrometry. Anal. Chem. 69,4197-4202 (1997), herein incorporated by reference in its entirety).This technique varies in how rapidly it can be performed, from a fewsamples per day to a high throughput of 40,000 SNPs per day, using masscode tags. A preferred example is the use of mass spectrometricdetermination of a nucleic acid sequence which includes thepolymorphisms of the invention, for example, which includes the promoterof the COX2 gene or a complementary sequence. Such mass spectrometricmethods are known to those skilled in the art, and the genotypingmethods of the invention are amenable to adaptation for the massspectrometric detection of the polymorphisms of the invention, forexample, the COX2 promoter polymorphisms of the invention.

SNPs can also be determined by ligation-bit analysis. This analysisrequires two primers that hybridize to a target with a one nucleotidegap between the primers. Each of the four nucleotides is added to aseparate reaction mixture containing DNA polymerase, ligase, target DNAand the primers. The polymerase adds a nucleotide to the 3′ end of thefirst primer that is complementary to the SNP, and the ligase thenligates the two adjacent primers together. Upon heating of the sample,if ligation has occurred, the now larger primer will remain hybridizedand a signal, for example, fluorescence, can be detected. A furtherdiscussion of these methods can be found in U.S. Pat. Nos. 5,919,626;5,945,283; 5,242,794; and 5,952,174 (each of the foregoing which isherein incorporated by reference in its entirety).

U.S. Pat. No. 6,821,733 (herein incorporated by reference in itsentirety) describes methods to detect differences in the sequence of twonucleic acid molecules that includes the steps of: contacting twonucleic acids under conditions that allow the formation of a four-waycomplex and branch migration; contacting the four-way complex with atracer molecule and a detection molecule under conditions in which thedetection molecule is capable of binding the tracer molecule or thefour-way complex; and determining binding of the tracer molecule to thedetection molecule before and after exposure to the four-way complex.Competition of the four-way complex with the tracer molecule for bindingto the detection molecule indicates a difference between the two nucleicacids.

Protein- and proteomics-based approaches are also suitable forpolymorphism detection and analysis. Polymorphisms which result in orare associated with variation in expressed proteins can be detecteddirectly by analysing said proteins. This typically requires separationof the various proteins within a sample, by, for example, gelelectrophoresis or HPLC, and identification of said proteins or peptidesderived therefrom, for example by NMR or protein sequencing such aschemical sequencing or more prevalently mass spectrometry. Proteomicmethodologies are well known in the art, and have great potential forautomation. For example, integrated systems, such as the ProteomIQ™system from Proteome Systems, provide high throughput platforms forproteome analysis combining sample preparation, protein separation,image acquisition and analysis, protein processing, mass spectrometryand bioinformatics technologies.

The majority of proteomic methods of protein identification utilise massspectrometry, including ion trap mass spectrometry, liquidchromatography (LC) and LC/MSn mass spectrometry, gas chromatography(GC) mass spectroscopy, Fourier transform-ion cyclotron resonance-massspectrometer (FT-MS), MALDI-TOF mass spectrometry, and ESI massspectrometry, and their derivatives. Mass spectrometric methods are alsouseful in the determination of post-translational modification ofproteins, such as phosphorylation or glycosylation, and thus haveutility in determining polymorphisms that result in or are associatedwith variation in post-translational modifications of proteins.

Associated technologies are also well known, and include, for example,protein processing devices such as the “Chemical Inkjet Printer”comprising piezoelectric printing technology that allows in situenzymatic or chemical digestion of protein samples electroblotted from2-D PAGE gels to membranes by jetting the enzyme or chemical directlyonto the selected protein spots (Sloane, A. J. et al. High throughputpeptide mass fingerprinting and protein macroarray analysis usingchemical printing strategies. Mol Cell Proteomics 1(7):490-9 (2002),herein incorporated by reference in its entirety). After in-situdigestion and incubation of the proteins, the membrane can be placeddirectly into the mass spectrometer for peptide analysis.

A large number of methods reliant on the conformational variability ofnucleic acids have been developed to detect SNPs.

For example, Single Strand Conformational Polymorphism (SSCP, Orita etal., PNAS 86:2766-2770 (1989), herein incorporated by reference in itsentirety) is a method reliant on the ability of single-stranded nucleicacids to form secondary structure in solution under certain conditions.The secondary structure depends on the base composition and can bealtered by a single nucleotide substitution, causing differences inelectrophoretic mobility under nondenaturing conditions. The variouspolymorphs are typically detected by autoradiography when radioactivelylabelled, by silver staining of bands, by hybridisation with detectablylabelled probe fragments or the use of fluorescent PCR primers which aresubsequently detected, for example by an automated DNA sequencer.

Modifications of SSCP are well known in the art, and include the use ofdiffering gel running conditions, such as for example differingtemperature, or the addition of additives, and different gel matrices.Other variations on SSCP are well known to the skilled artisan,including, RNA-SSCP (Gasparini, P. et al. Scanning the first part of theneurofibromatosis type 1 gene by RNA-SSCP: identification of three novelmutations and of two new polymorphisms. Hum Genet. 97(4):492-5 (1996),herein incorporated by reference in its entirety), restrictionendonuclease fingerprinting-SSCP (Liu, Q. et al. Restrictionendonuclease fingerprinting (REF): a sensitive method for screeningmutations in long, contiguous segments of DNA. Biotechniques 18(3):470-7(1995), herein incorporated by reference in its entirety), dideoxyfingerprinting (a hybrid between dideoxy sequencing and SSCP) (Sarkar,G. et al. Dideoxy fingerprinting (ddF): a rapid and efficient screen forthe presence of mutations. Genomics 13:441-443 (1992), hereinincorporated by reference in its entirety), bi-directional dideoxyfingerprinting (in which the dideoxy termination reaction is performedsimultaneously with two opposing primers) (Liu, Q. et al. Bi-directionaldideoxy fingerprinting (Bi-ddF): a rapid method for quantitativedetection of mutations in genomic regions of 300-600 bp. Hum Mol. Genet.5(1):107-14 (1996), herein incorporated by reference in its entirety),and Fluorescent PCR-SSCP (in which PCR products are internally labelledwith multiple fluorescent dyes, can be digested with restrictionenzymes, followed by SSCP, and analysed on an automated DNA sequencerable to detect the fluorescent dyes) (Makino, R. et al. F-SSCP:fluorescence-based polymerase chain reaction-single-strand conformationpolymorphism (PCR-SSCP) analysis. PCR Methods Appl. 2(1):10-13 (1992),herein incorporated by reference in its entirety).

Other methods which utilise the varying mobility of different nucleicacid structures include Denaturing Gradient Gel Electrophoresis (DGGE)(Cariello, N. F. et al. Resolution of a missense mutant in human genomicDNA by denaturing gradient gel electrophoresis and direct sequencingusing in vitro DNA amplification: HPRT Munich. Am J Hum Genet.42(5):726-34 (1988), herein incorporated by reference in its entirety),Temperature Gradient Gel Electrophoresis (TGGE) (Riesner, D. et al.Temperature-gradient gel electrophoresis for the detection ofpolymorphic DNA and for quantitative polymerase chain reaction.Electrophoresis. 13:632-6 (1992), herein incorporated by reference inits entirety), and Heteroduplex Analysis (HET) (Keen, J. et al. Rapiddetection of single base mismatches as heteroduplexes on Hydrolink gels.Trends Genet. 7(1):5 (1991), herein, incorporated by reference in itsentirety). Here, variation in the dissociation of double stranded DNA(for example, due to base-pair mismatches) results in a change inelectrophoretic mobility. These mobility shifts are used to detectnucleotide variations.

Denaturing High Pressure Liquid Chromatography (HPLC) is yet a furthermethod utilised to detect SNPs, using HPLC methods well-known in the artas an alternative to the separation methods described above (such as gelelectophoresis) to detect, for example, homoduplexes and heteroduplexeswhich elute from the HPLC column at different rates, thereby enablingdetection of mismatch nucleotides and thus SNPs (Giordano, M. et al.Identification by denaturing high-performance liquid chromatography ofnumerous polymorphisms in a candidate region for multiple sclerosissusceptibility. Genomics 56(3):247-53 (1999), herein incorporated byreference in its entirety).

Yet further methods to detect SNPs rely on the differing susceptibilityof single stranded and double stranded nucleic acids to cleavage byvarious agents, including chemical cleavage agents and nucleolyticenzymes. For example, cleavage of mismatches within RNA:DNAheteroduplexes by RNase A, of heteroduplexes by, for examplebacteriophage T4 endonuclease YII or T7 endonuclease I, of the 5′ end ofthe hairpin loops at the junction between single stranded and doublestranded DNA by cleavase I, and the modification of mispairednucleotides within heteroduplexes by chemical agents commonly used inMaxam-Gilbert sequencing chemistry, are all well known in the art.

Further examples include the Protein Translation Test (PTT), used toresolve stop codons generated by variations which lead to a prematuretermination of translation and to protein products of reduced size, andthe use of mismatch binding proteins (Moore, W. et al. Mutationdetection in the breast cancer gene BRCA1 using the protein truncationtest. Mol. Biotechnol. 14(2):89-97 (2000), herein incorporated byreference in its entirety). Variations are detected by binding of, forexample, the MutS protein, a component of Escherichia coli DNA mismatchrepair system, or the human hMSH2 and GTBP proteins, to double strandedDNA heteroduplexes containing mismatched bases. DNA duplexes are thenincubated with the mismatch binding protein, and variations are detectedby mobility shift assay. For example, a simple assay is based on thefact that the binding of the mismatch binding protein to theheteroduplex protects the heteroduplex from exonuclease degradation.

Those skilled in the art will know that a particular SNP, particularlywhen it occurs in a regulatory region of a gene such as a promoter, canbe associated with altered expression of a gene. Altered expression of agene can also result when the SNP is located in the coding region of aprotein-encoding gene, for example where the SNP is associated withcodons of varying usage and thus with tRNAs of differing abundance. Suchaltered expression can be determined by methods well known in the art,and can thereby be employed to detect such SNPs. Similarly, where a SNPoccurs in the coding region of a gene and results in a non-synonomousamino acid substitution, such substitution can result in a change in thefunction of the gene product. Similarly, in cases where the gene productis an RNA, such SNPs can result in a change of function in the RNA geneproduct. Any such change in function, for example as assessed in anactivity or functionality assay, can be employed to detect such SNPs.

The above methods of detecting and identifying SNPs are amenable to usein the methods of the invention.

In practicing the present invention to assess the risk a particularsubject faces with respect to a particular disease, that subject will beassessed to determine the presence or absence of polymorphisms(preferably SNPs) which are either associated with protection from thedisease or susceptibility to the disease.

In order to detect and identify SNPs in accordance with the invention, asample containing material to be tested is obtained from the subject.The sample can be any sample potentially containing the target SNPs (ortarget polypeptides, as the case may be) and obtained from any bodilyfluid (blood, urine, saliva, etc) biopsies or other tissue preparations.

DNA or RNA can be isolated from the sample according to any of a numberof methods well known in the art. For example, methods of purificationof nucleic acids are described in Tijssen; Laboratory Techniques inBiochemistry and Molecular Biology Hybridization with nucleic acidprobes Part 1: Theory and Nucleic acid preparation, Elsevier, New York,N.Y. 1993, as well as in Maniatis, T., Fritsch, E. F. and Sambrook, J.,Molecular Cloning Manual 1989 (each of the foregoing which is hereinincorporated by reference in its entirety).

Upon detection of the presence or absence of the polymorphisms testedfor, the critical step is to determine a net susceptibility score forthe subject. This score will represent the balance between the combinedvalue of the protective polymorphisms present and the total value of thesusceptibility polymorphisms present, with a net protective score (i.e.,a greater weight of protective polymorphisms present than susceptibilitypolymorphisms) being predictive of a reduced risk of developing thedisease in question. The reverse is true where there is a netsusceptibility score. To calculate where the balance lies, theindividual polymorphisms are assigned a value. In the simplestembodiment, each polymorphisms within a category (i.e. protective orsusceptibility) is assigned an equal value, with each protectivepolymorphism being −1 and each susceptibility polymorphism being +1 (orvice versa). It is however contemplated that the values assigned toindividual polymorphisms within a category can differ, with somepolymorphisms being assigned a value that reflects their predictive ordiscriminatory value. For example, one particularly strong protectivepolymorphism can have a value of −2, whereas another more weaklyprotective polymorphism can have a value of −0.75.

The net score, and the associated predictive outcome in terms of therisk of the subject developing a particular disease, can be representedin a number of ways. One example is as a graph as more particularlyexemplified herein.

Another example is a simple numerical score (eg +2 to represent asubject with a net susceptibility score or −2 to represent a subjectwith a net protective score). In each case, the result is communicatedto the subject with an explanation of what that result means to thatsubject. Preferably, advice on ways the subject can change theirlifestyle so as to reduce the risk of developing the disease is alsocommunicated to the subject.

It will be appreciated that the methods of the invention can beperformed in conjunction with an analysis of other risk factors known tobe associated with a disease, such as COPD, emphysema, OCOPD, or lungcancer. Such risk factors include epidemiological risk factorsassociated with an increased risk of developing the disease. Such riskfactors include, but are not limited to smoking and/or exposure totobacco smoke, age, sex and familial history. These risk factors can beused to augment an analysis of one or more polymorphisms as hereindescribed when assessing a subject's risk of developing a disease suchas COPD, emphysema, OCOPD, or lung cancer.

The predictive methods of the invention allow a number of therapeuticinterventions and/or treatment regimens to be assessed for suitabilityand implemented for a given subject, depending upon the disease and theoverall risk quotient. The simplest of these can be the provision to asubject with a net susceptibility score of motivation to implement alifestyle change, for example, in the case of OCOPD, to reduce exposureto aero-pollutants, for example, by an occupational change or by the useof safety equipment in the work place. Similarly where the subject is acurrent smoker, the methods of the invention can provide motivation toquit smoking. In this latter case, a ‘quit smoking’ program can befollowed, which can include the use of anti-smoking medicaments (such asnicotine patches and the like) as well as anti-addiction medicaments.

Other therapeutic interventions can involve altering the balance betweenprotective and susceptibility polymorphisms towards a protective state(such as by neutralizing or reversing a susceptibility polymorphism).The manner of therapeutic intervention or treatment will be predicatedby the nature of the polymorphism(s) and the biological effect of saidpolymorphism(s). For example, where a susceptibility polymorphism isassociated with a change in the expression of a gene, intervention ortreatment is preferably directed to the restoration of normal expressionof said gene, by, for example, administration of an agent capable ofmodulating the expression of said gene. Where a polymorphism, such as aSNP allele or genotype, is associated with decreased expression of agene, therapy can involve administration of an agent capable ofincreasing the expression of said gene, and conversely, where apolymorphism is associated with increased expression of a gene, therapycan involve administration of an agent capable of decreasing theexpression of said gene. Methods useful for the modulation of geneexpression are well known in the art. For example, in situations were apolymorphism is associated with upregulated expression of a gene,therapy utilising, for example, RNAi or antisense methodologies can beimplemented to decrease the abundance of mRNA and so decrease theexpression of said gene. Alternatively, therapy can involve methodsdirected to, for example, modulating the activity of the product of saidgene, thereby compensating for the abnormal expression of said gene.

Where a susceptibility polymorphism is associated with decreased geneproduct function or decreased levels of expression of a gene product,therapeutic intervention or treatment can involve augmenting orreplacing of said function, or supplementing the amount of gene productwithin the subject for example, by administration of said gene productor a functional analogue thereof. For example, where a polymorphism isassociated with decreased enzyme function, therapy can involveadministration of active enzyme or an enzyme analogue to the subject.Similarly, where a polymorphism is associated with increased geneproduct function, therapeutic intervention or treatment can involvereduction of said function, for example, by administration of aninhibitor of said gene product or an agent capable of decreasing thelevel of said gene product in the subject. For example, where apolymorphism is associated with increased enzyme function, therapy caninvolve administration of an enzyme inhibitor to the subject.

Likewise, when a protective polymorphism is associated with upregulationof a particular gene or expression of an enzyme or other protein,therapies can be directed to mimic such upregulation or expression in anindividual lacking the resistive genotype, and/or delivery of suchenzyme or other protein to such individual Further, when a protectivepolymorphism is associated with downregulation of a particular gene, orwith diminished or eliminated expression of an enzyme or other protein,desirable therapies can be directed to mimicking such conditions in anindividual that lacks the protective genotype.

EXAMPLES

The invention will now be described in more detail, with reference tonon-limiting examples.

Example 1 Case Association Study—COPD Methods Subject Recruitment

Subjects of European descent who had smoked a minimum of fifteen packyears and diagnosed by a physician with chronic obstructive pulmonarydisease (COPD) were recruited. Subjects met the following criteria: wereover 50 years old and had developed symptoms of breathlessness after 40years of age, had a Forced expiratory volume in one second (FEV1) as apercentage of predicted <70% and a FEV1/FVC ratio (Forced expiratoryvolume in one second/Forced vital capacity) of <79% (measured usingAmerican Thoracic Society criteria). Two hundred and ninety-foursubjects were recruited, of these 58% were male, the mean FEV1/FVC (±95%confidence limits) was 51% (49-53), mean FEV1 as a percentage ofpredicted was 43 (41-45). Mean age, cigarettes per day and pack yearhistory was 65 yrs (64-66), 24 cigarettes/day (22-25) and 50 pack years(41-55) respectively. Two hundred and seventeen European subjects whohad smoked a minimum of twenty pack years and who had never sufferedbreathlessness and had not been diagnosed with an obstructive lungdisease in the past, in particular childhood asthma or chronicobstructive lung disease, were also studied. This control group wasrecruited through clubs for the elderly and consisted of 63% male, themean FEV1/FVC (95% CI) was 82% (81-83), mean FEV1 as a percentage ofpredicted was 96 (95-97). Mean age, cigarettes per day and pack yearhistory was 59 yrs (57-61), 24 cigarettes/day (22-26) and 42 pack years(39-45) respectively. Using a PCR based method [1, incorporated hereinin its entirety by reference], all subjects were genotyped for theα1-antitrypsin mutations (S and Z alleles) and those with the ZZ allelewere excluded. The COPD and resistant smoker cohorts were matched forsubjects with the MZ genotype (5% in each cohort). 190 European blooddonors (smoking status unknown) were recruited consecutively throughlocal blood donor services. Sixty-three percent were men and their meanage was 50 years. On regression analysis, the age difference and packyears difference observed between COPD sufferers and resistant smokerswas found not to determine FEV or COPD.

This study shows that polymorphisms found in greater frequency in COPDpatients compared to controls (and/or resistant smokers) can reflect anincreased susceptibility to the development of impaired lung functionand COPD. Similarly, polymorphisms found in greater frequency inresistant smokers compared to susceptible smokers (COPD patients and/orcontrols) can reflect a protective role.

Summary of Characteristics for the COPD, Resistant Smoker and HealthyBlood Donors

Parameter COPD Resistant smokers Median (IQR) N = 294 N = 217Differences % male 58% 63% ns Age (yrs) 65 (64-66) 59 (57-61) P < 0.05Pack years 50 (46-53) 42 (39-45) P < 0.05 Cigarettes/day 24 (22-25) 24(22-26) ns FEV1 (L)  1.6 (0.7-2.5)  2.9 (2.8-3.0) P < 0.05 FEV1 %predict 43 (41-45) 96% (95-97)   P < 0.05 FEV1/FVC 51 (49-53) 82 (81-83)P < 0.05Means and 95% confidence limitsCyclo-oxygenase 2 (COX2) −765 G/C Promoter Polymorphism anda1-antitrypsin Genotyping

Genomic DNA was extracted from whole blood samples [2, hereinincorporated by reference in its entirety]. The Cyclo-oxygenase 2 −765polymorphism was determined by minor modifications of a previouslypublished method [3, herein incorporated by reference in its entirety].The PCR reaction was carried out in a total volume of 25 ul andcontained 20 ng genomic DNA, 500 pmol forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.0 mM MgCl₂ and 1 unitof polymerase (Life Technologies). Cycling times were incubations for 3min at 95° C. followed by 33 cycles of 50 s at 94° C., 60 s at 66° C.and 60 s at 72° C. A final elongation of 10 min at 72° C. then followed.4 ul of PCR products were visualised by ultraviolet trans-illuminationof a 3% agarose gel stained with ethidium bromide. An aliquot of 3 ul ofamplification product was digested for 1 hr with 4 units of AciI (RocheDiagnostics, New Zealand) at 37° C. Digested products were separated ona 2.5% agarose gel run for 2.0 hours at 80 mV with TBE buffer. Theproducts were visualised against a 123 bp ladder using ultraviolettransillumination after ethidium bromide staining. Using a PCR basedmethod referenced above [1, herein incorporated by reference in itsentirety], all COPD and resistant smoker subjects were genotyped for theα1-antitrypsin S and Z alleles.

Other Polymorphism Genotyping

Genomic DNA was extracted from whole blood samples [2]. Purified genomicDNA was aliquoted (10 ng/ul concentration) into 96 well plates andgenotyped on a Sequenom™ system (Sequenom™ Autoflex Mass Spectrometerand Samsung 24 pin nanodispenser) using the following sequences,amplification conditions and methods.

The following conditions were used for the PCR multiplex reaction: finalconcentrations were for 10× Buffer 15 mM MgCl₂ 1.25×, 25 mM MgCl₂ 1.625mM, dNTP mix 25 mM 500 uM, primers 4 uM 100 nM, Taq polymerase (Qiagenhot start) 0.15 U/reaction, Genomic DNA 10 ng/ul. Cycling times were 95°C. for 15 mM, (5° C. for 15 s, 56° C. 30 s, 72° C. 30 s for 45 cycleswith a prolonged extension time of 3 min to finish. Shrimp alkalinephosphotase (SAP) treatment was used (2 ul to 5 ul per PCR reaction)incubated at 35° C. for 30 min and extension reaction (add 2 ul to 7 ulafter SAP treatment) with the following volumes per reaction of: water,0.76 ul; hME 10× termination buffer, 0.2 ul; hME primer (10 uM), 1 ul;MassEXTEND enzyme, 0.04 ul.

Sequenom Conditions for the Polymorphisms Genotyping—1

SNP_ID TERM WELL 2nd-PCRP 1st-PCRP Vitamin ACT W1ACGTTGGATGGCTTGTTAACCAGCTTTGCC ACGTTGGATGTTTTTCAGACTGGCAGAGCG [SEQ. ID.NO. 2] DBP-420 [SEQ. ID. NO. 1] Vitamin ACT W1ACGTTGGATGTTTTTCAGACTGGCAGAGCG ACGTTGGATGGCTTGTTAACCAGCTTTGCC [SEQ. ID.NO. 4] DBP-416 [SEQ. ID. NO. 3] IL13 C- ACT W2ACGTTGGATGCATGTCGCCTTTTCCTGCTC ACGTTGGATGCAACACCCAACAGGCAAATG [SEQ. ID.NO. 6] 1055T [SEQ. ID. NO. 5] GSTP1- ACT W2ACGTTGGATGTGGTGGACATGGTGAATGAC ACGTTGGATGTGGTGCAGATGCTCACATAG [SEQ. ID.NO. 8] 105 [SEQ. ID. NO. 7] PAI1 G- ACT W2ACGTTGGATGCACAGAGAGAGTCTGGACAC ACGTTGGATGCTCTTGGTCTTTCCCTCATC [SEQ. ID.NO. 10] 675G [SEQ. ID. NO. 9] NOS3- ACT W3ACGTTGGATGACAGCTCTGCATTCAGCACG ACGTTGGATGAGTCAATCCCTTTGGTGCTC [SEQ. ID.NO. 12] 298 [SEQ. ID. NO. 11] IL13- ACT W3ACGTTGGATGGTTTTCCAGCTTGCATGTCC ACGTTGGATGCAATAGTCAGGTCCTGTCTC [SEQ. ID.NO. 14] Arg130Gln [SEQ. ID. NO. 13] ADRB2- ACT W3ACGTTGGATGGAACGGCAGCGCCTTCTTG ACGTTGGATGACTTGGCAATGGCTGTGATG [SEQ. ID.NO. 16] Arg16Gly [SEQ. ID. NO. 15] IFNG- CGT W5ACGTTGGATGCAGACATTCACAATTGATTT ACGTTGGATGGATAGTTCCAAACATGTGCG [SEQ. ID.NO. 18] A874T [SEQ. ID. NO. 17] IL18-C- ACT W6ACGTTGGATGGGGTATTCATAAGCTGAAAC ACGTTGGATGCCTTCAAGTTCAGTGGTCAG [SEQ. ID.NO. 20] 133G [SEQ. ID. NO. 19] IL18- ACT W8ACGTTGGATGGGTCAATGAAGAGAACTTGG ACGTTGGATGAATGTTTATTGTAGAAAACC [SEQ. ID.NO. 22] A105C [SEQ. ID. NO. 21]

Sequenom Conditions for the Polymorphisms Genotyping—2

SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC PWARN UEP_DIR Vitamin DBP -420 99 99.7 99.7 46.2 53.3 ML R Vitamin DBP - 416 99 99.7 99.7 45.5 33.3M F IL13 C-1055T 112 97.5 80 48.2 60 L R GSTP1 - 105 107 99.4 80 49.952.9 F PAI1 G-675G 109 97.9 80 59.3 66.7 g F NOS3 -298 186 98.1 65 61.263.2 F IL13-Arg130Gln 171 99.3 65 55.1 47.6 F ADRB2- Arg16Gly 187 88.265 65.1 58.3 F IFNG - A874T 112 75.3 81.2 45.6 27.3 F IL18- C-133G 11293.5 74.3 41.8 46.7 L F IL18- A105C 121 67.2 74.3 48.9 40 R

Sequenom Conditions for the Polymorphisms Genotyping—3

SNP_ID UEP_MASS UEP_SEQ EXT1_CALL EXT1_MASS Vitamin DBP - 420 4518.9AGCTTTGCCAGTTCC [SEQ. ID. NO. 23] A 4807.1 Vitamin DBP - 416 5524.6AAAAGCAAAATTGCCTGA [SEQ. ID. NO. 24] T 5812.8 IL13 C-1055T 4405.9TCCTGCTCTTCCCTC [SEQ. ID. NO. 25] T 4703.1 GSTP1 - 105 5099.3ACCTCCGCTGCAAATAC [SEQ. ID. NO. 26] A 5396.5 PAI1 G-675G 5620.6GAGTCTGGACACGTGGGG [SEQ. ID. NO. 27] DEL 5917.9 NOS3 -298 5813.8TGCTGCAGGCCCCAGATGA [SEQ. ID. NO. 28] T 6102 IL13-Arg130Gln 6470.2AGAAACTTTTTCGCGAGGGAC [SEQ. ID. NO. 29] A 6767.4 ADRB2- Arg16Gly 7264.7AGCGCCTTCTTGCTGGCACCCAAT [SEQ. ID. NO. 30] A 7561.9 IFNG - A874T 6639.4TCTTACAACACAAAATCAAATC [SEQ. ID. NO. 31] T 6927.6 IL18- C-133G 4592AGCTGAAACTTCTGG [SEQ. ID. NO. 32] C 4865.2 IL18- A105C 6085TCAAGCTTGCCAAAGTAATC [SEQ. ID. NO. 33] A 6373.2

Sequenom Conditions for the Polymorphisms Genotyping—4

SNP_ID EXT1_SEQ EXT2_CALL EXT2_MASS EXT2_SEQ 1stPAUSE VitaminAGCTTTGCCAGTTCCT [SEQ. ID. NO. 34] C 5136.4 AGCTTTGCCAGTTCCGT 4848.2DBP - 420 [SEQ. ID. NO. 35] Vitamin AAAAGCAAAATTGCCTGAT [SEQ. ID. NO.36] G 6456.2 AAAAGCAAAATTGCCTGAGGC 5853.9 DBP-416 [SEQ. ID. NO. 37] IL13C- TCCTGCTCTTCCCTCA [SEQ. ID. NO. 38] C 5023.3 TCCTGCTCTTCCCTCGT 4735.11055T [SEQ. ID. NO. 39] GSTP1- ACCTCCGCTGCAAATACA [SEQ. ID. NO. 40] G5716.7 ACCTCCGCTGCAAATACGT 5428.5 105 [SEQ. ID. NO. 41] PAI1 G-GAGTCTGGACACGTGGGGA [SEQ. ID. NO. 42] G 6247.1 GAGTCTGGACACGTGGGGGA5949.9 675G [SEQ. ID. NO. 43] NOS3- TGCTGCAGGCCCCAGATGAT [SEQ. ID. NO. G6416.2 TGCTGCAGGCCCCAGATGAGC 6143 298 44] [SEQ. ID. NO. 45] IL13-AGAAACTTTTTCGCGAGGGACA G 7416.8 AGAAACTTTTTCGCGAGGGACGGT 6799.4Arg130Gln [SEQ. ID. NO. 46] [SEQ. ID. NO. 47] ADRB2-AGCGCCTTCTTGCTGGCACCCAATA G 8220.3 AGCGCCTTCTTGCTGGCACCCAATGGA 7593.9Arg16Gly [SEQ. ID. NO. 48] [SEQ. ID. NO. 49] IFNG-TCTTACAACACAAAATCAAATCT A 7225.8 TCTTACAACACAAAATCAAATCAC 6952.6 A874T[SEQ. ID. NO. 50] [SEQ. ID. NO. 51] IL18- C- AGCTGAAACTTCTGGC [SEQ. ID.NO. 52] G 5218.4 AGCTGAAACTTCTGGGA 4921.2 133G [SEQ. ID. NO. 53] IL18-TCAAGCTTGCCAAAGTAATCT C 7040.6 TCAAGCTTGCCAAAGTAATCGGA 6414.2 A105C[SEQ. ID. NO. 54] [SEQ. ID. NO. 55]

Sequenom Conditions for the Polymorphisms Genotyping—5

SNP_ID 2nd-PCRP 1st-PCRP Lipoxygenase5- ACGTTGGATGGAAGTCAGAGATGATGGCAGACGTTGGATGATGAATCCTGGACCCAAGAC 366G/A [SEQ. ID. NO. 56] [SEQ. ID. NO.57] TNFalpha+489G/A ACGTTGGATGGAAAGATGTGCGCTGATAGGACGTTGGATGGCCACATCTCTTTCTGCATC [SEQ. ID. NO. 58] [SEQ. ID. NO. 59]SMAD3C89Y ACGTTGGATGTTGCAGGTGTCCCATCGGAA [SEQ. ID. NO. 60]ACGTTGGATGTAGCTCGTGGTGGCTGTGCA [SEQ. ID. NO. 61] CaspaseGly881ArgG/CACGTTGGATGGTGATCACCCAAGGCTTCAG [SEQ. ID. NO. 62]ACGTTGGATGGTCTGTTGACTCTTTTGGCC [SEQ. ID. NO. 63] MBL2+161G/AACGTTGGATGGTAGCTCTCCAGGCATCAAC [SEQ. ID. NO. 64]ACGTTGGATGGTACCTGGTTCCCCCTTTTC [SEQ. ID. NO. 65] HSP70-HOM2437T/CACGTTGGATGTGATCTTGTTCACCTTGCCG [SEQ. ID. NO. 66]ACGTTGGATGAGATCGAGGTGACGTTTGAC [SEQ. ID. NO. 67] CD14-159C/TACGTTGGATGAGACACAGAACCCTAGATGC [SEQ. ID. NO. 68]ACGTTGGATGGCAATGAAGGATGTTTCAGG [SEQ. ID. NO. 69] Chymase1-1903G/AACGTTGGATGTAAGACAGCTCCACAGCATC [SEQ. ID. NO. 70]ACGTTGGATGTTCCATTTCCTCACCCTCAG [SEQ. ID. NO. 71] TNFalpha-308G/AACGTTGGATGGATTTGTGTGTAGGACCCTG [SEQ. ID. NO. 72]ACGTTGGATGGGTCCCCAAAAGAAATGGAG [SEQ. ID. NO. 73] CLCA1+13924T/AACGTTGGATGGGATTGGAGAACAAACTCAC [SEQ. ID. NO. 74]ACGTTGGATGGGCAGCTGTTACACCAAAAG [SEQ. ID. NO. 75] MEHTyr113HisT/CACGTTGGATGCTGGCGTTTTGCAAACATAC [SEQ. ID. NO. 76]ACGTTGGATGTTGACTGGAAGAAGCAGGTG [SEQ. ID. NO. 77] NAT2Arg197GlnG/AACGTTGGATGCCTGCCAAAGAAGAAACACC [SEQ. ID. NO. 78]ACGTTGGATGACGTCTGCAGGTATGTATTC [SEQ. ID. NO. 79] MEHHis139ArgG/AACGTTGGATGACTTCATCCACGTGAAGCCC [SEQ. ID. NO. 80]ACGTTGGATGAAACTCGTAGAAAGAGCCGG [SEQ. ID. NO. 81] IL-1B-511A/GACGTTGGATGATTTTCTCCTCAGAGGCTCC [SEQ. ID. NO. 82]ACGTTGGATGTGTCTGTATTGAGGGTGTGG [SEQ. ID. NO. 83] ADRB2Gln27GluC/GACGTTGGATGTTGCTGGCACCCAATGGAAG [SEQ. ID. NO. 84]ACGTTGGATGATGAGAGACATGACGATGCC [SEQ. ID. NO. 85] ICAM1E469KA/GACGTTGGATGACTCACAGAGCACATTCACG [SEQ. ID. NO. 86]ACGTTGGATGTGTCACTCGAGATCTTGAGG [SEQ. ID. NO. 87]

Sequenom Conditions for the Polymorphisms Genotyping—6

SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC UEP_DIR Lipoxygenase5-366G/A104 99.6 73.4 59 70.6 F TNFalpha + 489G/A 96 99.6 73.4 45.5 38.9 FSMAD3C89Y 107 87.3 71.7 45.7 47.1 F CaspaseGly881ArgG/C 111 97.2 81 52.958.8 R MBL2 + 161G/A 99 96.8 81 50.3 52.9 F HSP70-HOM2437T/C 107 99.3 8162.2 65 R CD14-159C/T 92 98 76.7 53.3 50 F Chymase1-1903G/A 105 99.676.7 53.6 39.1 R TNFalpha-308G/A 100 99.7 81.6 59.9 70.6 R CLCA1 +13924T/A 101 98 98 45.3 36.8 R MEHTyr113HisT/C 103 97.7 82.2 48.7 42.1 RNAT2Arg197GlnG/A 115 97.4 70 48.5 36.4 F MEHHis139ArgG/A 115 96.7 77.866 82.4 F IL-1B-511A/G 111 99.2 83 46 47.1 R ADRB2Gln27GluC/G 118 96.680 52.2 66.7 F ICAM1E469KA/G 115 98.8 95.8 51.5 52.9 R

Sequenom Conditions for the Polymorphisms Genotyping—7

SNP_ID UEP_MASS UEP_SEQ EXT1_CALL EXT1_MASS Lipoxygenase5-366G/A 5209.4GTGCCTGTGCTGGGCTC [SEQ. ID. NO. 88] A 5506.6 TNFalpha+489G/A 5638.7GGATGGAGAGAAAAAAAC [SEQ. ID. NO. 89] A 5935.9 SMAD3C89Y 5056.3CCCTCATGTCATCTACT [SEQ. ID. NO. 90] A 5353.5 CaspaseGly881ArgG/C 5097.3GTCACCCACTCTGTTGC [SEQ. ID. NO. 91] G 5370.5 MBL2+161G/A 5299.5CAAAGATGGGCGTGATG [SEQ. ID. NO. 92] A 5596.7 HSP70-HOM2437T/C 6026.9CCTTGCCGGTGCTCTTGTCC [SEQ. ID. NO. 93] T 6324.1 CD14-159C/T 6068CAGAATCCTTCCTGTTACGG [SEQ. ID. NO. 94] C 6341.1 Chymase1-1903G/A 6973.6TCCACCAAGACTTAAGTTTTGCT [SEQ. ID. NO. 95] G 7246.7 TNFalpha-308G/A5156.4 GAGGCTGAACCCCGTCC [SEQ. ID. NO. 96] G 5429.5 CLCA1+13924T/A5759.8 CTTTTTCATAGAGTCCTGT [SEQ. ID. NO. 97] A 6048 MEHTyr113HisT/C5913.9 TTAGTCTTGAAGTGAGGGT [SEQ. ID. NO. 98] T 6211.1 NAT2Arg197GlnG/A6635.3 TACTTATTTACGCTTGAACCTC [SEQ. ID. NO. 99] A 6932.5 MEHHis139ArgG/A5117.3 CCAGCTGCCCGCAGGCC [SEQ. ID. NO. 100] A 5414.5 IL-1B-511A/G 5203.4AATTGACAGAGAGCTCC [SEQ. ID. NO. 101] G 5476.6 ADRB2Gln27GluC/G 4547CACGACGTCACGCAG [SEQ. ID. NO. 102] C 4820.2 ICAM1E469KA/G 5090.3CACATTCACGGTCACCT [SEQ. ID. NO. 103] G 5363.5

Sequenom Conditions for the Polymorphisms Genotyping—8

EXT2 EXT2 1^(st) SNP_ID EXT1_SEQ CALL MASS EXT2_SEQ PAUSE Lipoxygenase5-GTGCCTGTGCTGGGCTCA G 5826.8 GTGCCTGTGCTGGGCTCGT [SEQ. ID. NO. 105]5538.6 366G/A [SEQ. ID. NO. 104] TNFalpha+489G/A GGATGGAGAGAAAAAAACA G6256.1 GGATGGAGAGAAAAAAACGT [SEQ. ID. NO. 107] 5967.9 [SEQ. ID. NO. 106]SMAD3C89Y CCCTCATGTCATCTACTA G 5658.7 CCCTCATGTCATCTACTGC [SEQ. ID. NO.109] 5385.5 [SEQ. ID. NO. 108] CaspaseGly881ArgG/C GTCACCCACTCTGTTGCC C5699.7 GTCACCCACTCTGTTGCGC [SEQ. ID. NO. 111] 5426.5 [SEQ. ID. NO. 110]MBL2+161G/A CAAAGATGGGCGTGATGA G 5901.9 CAAAGATGGGCGTGATGGC [SEQ. ID.NO. 113] 5628.7 [SEQ. ID. NO. 112] HSP70-HOM2437T/CCCTTGCCGGTGCTCTTGTCCA C 6644.3 CCTTGCCGGTGCTCTTGTCCGT 6356.1 [SEQ. ID.NO. 114] [SEQ. ID. NO. 115] CD14-159C/T CAGAATCCTTCCTGTTACGGC T 6645.3CAGAATCCTTCCTGTTACGGTC [SEQ. ID. NO. 117] 6372.2 [SEQ. ID. NO. 116]Chymase1-1903G/A TCCACCAAGACTTAAGTTTTGCTC A 7550.9TCCACCAAGACTTAAGTTTTGCTTC 7277.8 [SEQ. ID. NO. 118] [SEQ. ID. NO. 119]TNFalpha-308G/A GAGGCTGAACCCCGTCCC A 5733.7 GAGGCTGAACCCCGTCCTC [SEQ.ID. NO. 121] 5460.6 [SEQ. ID. NO. 120] CLCA1+13924T/ACTTTTTCATAGAGTCCTGTT T 6659.4 CTTTTTCATAGAGTCCTGTAAC [SEQ. ID. NO. 123]6073 [SEQ. ID. NO. 122] MEHTyr113HisT/C TTAGTCTTGAAGTGAGGGTA C 6531.3TTAGTCTTGAAGTGAGGGTGT [SEQ. ID. NO. 125] 6243.1 [SEQ. ID. NO. 124]NAT2Arg197GlnG/A TACTTATTTACGCTTGAACCTCA G 7261.8TACTTATTTACGCTTGAACCTCGA 6964.5 [SEQ. ID. NO. 126] [SEQ. ID. NO. 127]MEHHis139ArgG/A CCAGCTGCCCGCAGGCCA G 5734.7 CCAGCTGCCCGCAGGCCGT [SEQ.ID. NO. 129] 5446.5 [SEQ. ID. NO. 128] IL-1B-511A/G AATTGACAGAGAGCTCCC A5820.8 AATTGACAGAGAGCTCCTG [SEQ. ID. NO. 131] 5507.6 [SEQ. ID. NO. 130]ADRB2Gln27GluC/G CACGACGTCACGCAGC G 5173.4 CACGACGTCACGCAGGA [SEQ. ID.NO. 133] 4876.2 [SEQ. ID. NO. 132] ICAM1E469KA/G CACATTCACGGTCACCTC A5707.7 CACATTCACGGTCACCTTG [SEQ. ID. NO. 135] 5394.5 [SEQ. ID. NO. 134]

Results

Frequencies of individual polymorphisms are as follows:

TABLE 1 Polymorphism allele and genotype frequencies in the COPDpatients and resistant smokers. Cyclo-oxygenase 2 −765 G/C Allele*Genotype Frequency C G CC CG GG Controls n = 94 (%) 27 (14%) 161 (86%) 3 (3%) 21 (22%)  70 (75%) COPD n = 202 (%) 59 (15%) 345 (85%)  6 (3%)47 (23%) 149 (74%) Resistant n = 172 (%) 85² (25%)  259 (75%) 14¹ (8%) 57 (33%) 101 (59%) Beta2-adrenoreceptor Arg 16 Gly Allele* GenotypeFrequency A G AA AG GG Controls n = 182 (%) 152 (42%) 212 (58%) 26 (14%)100 (55%)   56 (31%) COPD n = 236 (%) 164 (34%) 308 (66%) 34 (14%)  96(41%) 106³ (45%) Resistant n = 190 (%) 135 (36%) 245 (64%) 34 (18%)  67(35%)  89⁴ (47%) Interleukin 18 105 A/C Allele* Genotype Frequency C ACC AC AA Controls n = 184 (%) 118 (32%) 250 (68%)  22 (12%) 74 (40%)   88 (48%) COPD n = 240 (%) 122 (25%) 377⁶ (75%)  21 (9%) 80 (33%)139^(5, 7) (58%) Resistant n = 196 (%) 113 (29%) 277 (71%) 16 (8%) 81(41%)    99 (50%) Interleukin 18 −133 C/G Allele* Genotype Frequency G CGG GC CC Controls n = 187 (%) 120 (32%) 254 (68%)  23 (12%) 74 (40%)  90(48%) COPD n = 238 123 (26%) 353⁹ (74%)  21 (9%) 81 (34%) 136⁸ (57%) Resistant n = 195 (%) 113 (29%) 277 (71%) 16 (8%) 81 (42%)  98 (50%)Plasminogen activator inhibitor 1 −675 4G/5G Allele* Genotype Frequency5G 4G 5G5G 5G4G 4G4G Controls n = 186 (%)  158 (42%) 214 (58%)    31(17%)  96 (52%)    59 (32%) COPD n = 237 (%) 219¹² (46%) 255 (54%)54^(10,11) (23%) 111 (47%)    72 (30%) Resistant n = 194 (%)  152 (39%)236 (61%)    31 (16%)  90 (46%) 73^(10,11) (38%) Nitric oxide synthase 3Asp 298 Glu (T/G) Allele* Genotype Frequency T G TT TG GG Controls n =183 (%) 108 (30%) 258 (70%)  13 (7%)   82 (45%)  88 (48%) COPD n = 238(%) 159 (42%) 317 (58%)  25 (10%) 109 (47%) 104 (43%) Resistant n = 194(%) 136 (35%) 252 (65%) 28¹³ (15%)  80 (41%)  86 (44%) Vitamin D BindingProtein Lys 420 Thr (A/C) Allele* Genotype Frequency A C AA AC CCControls n = 189 (%)  113 (30%) 265 (70%)  17 (9%)   79 (42%)  93 (49%)COPD n = 250 (%)  147 (29%) 353 (71%)  24 (10%)  99 (40%) 127 (50%)Resistant n = 195 (%) 140¹⁵ (36%) 250 (64%) 25¹⁴ (13%) 90¹⁴ (46%)  80(41%) Vitamin D Binding Protein Glu 416 Asp (T/G) Allele* GenotypeFrequency T G TT TG GG Controls n = 188 (%)  162 (43%) 214 (57%)  35(19%)   92 (49%) 61 (32%) COPD n = 240 (%)  230 (48%) 250 (52%)  57(24%)  116 (48%) 67 (28%) Resistant n = 197 (%) 193¹⁷ (49%) 201 (51%)43¹⁶ (22%) 107¹⁶ (54%) 47 (24%) Glutathione S Transferase P1 Ile 105 Val(A/G) Allele* Genotype Frequency A G AA AG GG Controls n = 185 (%)  232(63%) 138 (37%)  70 (38%)  92 (50%) 23 (12%) COPD n = 238 (%)  310 (65%)166 (35%)  96 (40%) 118 (50%) 24 (10%) Resistant n = 194 (%) 269¹⁹ (69%)119 (31%) 91¹⁸ (47%)  87 (45%) 16 (8%)  Interferon-gamma 874 A/T Allele*Genotype Frequency A T AA AT TT Controls n = 186 (%) 183 (49%) 189 (51%) 37 (20%) 109 (58%) 40 (22%) COPD n = 235 (%) 244 (52%) 226 (48%) 64²⁰(27%) 116 (49%) 55 (24%) Resistant n = 193 (%) 208 (54%) 178 (46%)  51(27%) 106 (55%) 36 (18%) Interleukin-13 Arg 130 Gln (G/A) Allele*Genotype Frequency A G AA AG GG Controls n = 184 (%) 67 (18%) 301 (82%) 3 (2%) 61 (33%) 120 (65%) COPD n = 237 (%) 86 (18%) 388 (82%)  8 (3%)70 (30%) 159 (67%) Resistant n = 194 (%) 74 (19%) 314 (81%) 9²¹ (5%) 56(28%) 129 (67%) Interleukin-13 −1055 C/T Allele* Genotype Frequency T CTT TC CC Controls n = 182 (%) 65 (18%) 299 (82%)  5 (3%) 55 (30%) 122(67%) COPD n = 234 (%) 94 (20%) 374 (80%) 8²² (4%) 78 (33%) 148 (63%)Resistant n = 192 (%) 72 (19%) 312 (81%)  2 (1%) 68 (35%) 122 (64%)a1-antitrypsin S Allele* Genotype Frequency M S MM MS SS COPD n = 202(%) 391 (97%) 13 (3%) 189 (94%)  13 (6%)   0 (0%) Resistant n = 189 (%)350 (93%) 28 (7%) 162 (85%) 26²³ (14%) 1²³ (1%) *number of chromosomes(2n)Genotype

-   -   1. Genotype. CC/CG vs GG for resistant vs COPD, Odds ratio        (OR)=1.98, 95% confidence limits 1.3-3.1, χ² (Yates        corrected)=8.82, p=0.003, CC/CG=protective for COPD    -   2. Allele. C vs G for resistant vs COPD, Odds ratio (OR)=1.92,        95% confidence limits 1.3-2.8, χ² (Yates corrected)=11.56,        p<0.001, C=protective for COPD    -   3. Genotype. GG vs AG/AA for COPD vs controls, Odds ratio        (OR)=1.83, 95% confidence limits 1.2-2.8, χ2 (Yates        corrected)=8.1, p=0.004, GG=susceptible to COPD (depending on        the presence of other snps)    -   4. Genotype. GG vs AG/AA for resistant vs controls, Odds ratio        (OR)=1.98, 95% confidence limits 1.3-3.1, χ2 (Yates        corrected)=9.43, p=0.002 GG=resistance (depending on the        presence of other snps)    -   5. Genotype. AA vs AC/CC for COPD vs controls, Odds ratio        (OR)=1.50, 95% confidence limits 1.0-2.3, χ2 (Yates        uncorrected)=4.26, p=0.04, AA=susceptible to COPD    -   6. Allele. A vs C for COPD vs control, Odds ratio (OR)=1.46, 95%        confidence limits 1.1-2.0, χ2 (Yates corrected)=5.76, p=0.02    -   7. Genotype. AA vs AC/CC for COPD vs resistant, Odds ratio        (OR)=1.35, 95% confidence limits 0.9-2.0, χ2 (Yates        uncorrected)=2.39, p=0.12 (trend),        -   AA=susceptible to COPD    -   8. Genotype. CC vs CG/GG for COPD vs controls, Odds ratio        (OR)=1.44, 95% confidence limits 1.0-2.2, χ2 (Yates        corrected)=3.4, p=0.06, CC=susceptible to COPD    -   9. Allele. C vs G for COPD vs control, Odds ratio (OR)=1.36, 95%        confidence limits 1.0-1.9, χ2 (Yates corrected)=53.7, p=0.05,        C=susceptible to COPD    -   10. Genotype. 5G5G vs rest for COPD vs resistant, Odds ratio        (OR)=1.55, 95% confidence limits 0.9-2.6, χ2 (Yates        uncorrected)=3.12, p=0.08, 5G5G=susceptible to COPD    -   11. Genotype. 5G5G vs rest for COPD vs control, Odds ratio        (OR)=1.48, 95% confidence limits 0.9-2.5, χ2 (Yates        uncorrected)=2.43, p=0.12, 5G5G=susceptible to COPD    -   12. Allele. 5G vs 4G for COPD vs resistant, Odds ratio        (OR)=1.33, 95% confidence limits 1.0-1.8, χ2 (Yates        corrected)=4.02, p=0.05, 5G=susceptible to COPD    -   13. Genotype. TT vs TG/GG for resistant vs controls, Odds ratio        (OR)=2.2, 95% confidence limits 1.0-4.7, χ2 (Yates        corrected)=4.49, p=0.03, TT genotype=protective for COPD    -   14. Genotype. AA/AC vs CC for resistant vs COPD, Odds ratio        (OR)=1.39, 95% confidence limits 0.9-2.1, χ2 (Yates        uncorrected)=2.59, p=0.10, AA/AC genotype=protective for COPD    -   15. Allele. A vs C for resistant vs COPD, Odds ratio (OR)=1.34,        95% confidence limits 1.0-1.8, χ2 (Yates corrected)=3.94,        p=0.05, A allele=protective for COPD    -   16. Genotype. TT/TG vs GG for resistant vs controls, Odds ratio        (OR)=1.53, 95% confidence limits 1.0-2.5, χ2 (Yates        uncorrected)=3.52, p=0.06, TT/TG genotype=protective for COPD    -   17. Allele. T vs G for resistant vs control, Odds ratio        (OR)=1.27, 95% confidence limits 1.0-1.7, χ2 (Yates        corrected)=2.69, p=0.1, T allele=protective for COPD    -   18. Genotype. AA vs AG/GG for resistant vs controls, Odds ratio        (OR)=1.45, 95% confidence limits 0.9-2.2, χ2 (Yates        uncorrected)=3.19, p=0.07, AA genotype=protective for COPD    -   19. Allele. A vs G for resistant vs control, Odds ratio        (OR)=1.34, 95% confidence limits 1.0-1.8, χ2 (Yates        uncorrected)=3.71, p=0.05, A allele=protective for COPD    -   20. Genotype. AA vs AT/TT for COPD vs controls, Odds ratio        (OR)=1.51, 95% confidence limits 0.9-2.5, χ2 (Yates        uncorrected)=3.07, p=0.08, AA genotype=susceptible to COPD    -   21. Genotype. AA vs AG/GG for resistant vs controls, Odds ratio        (OR)=2.94, 95% confidence limits 0.7-14.0, χ2 (Yates        uncorrected)=2.78, p=0.09, AA genotype=protective for COPD    -   22. Genotype. TT vs TC/CC for COPD vs resistant, Odds ratio        (OR)=6.03, 95% confidence limits 1.1-42, χ2 (Yates        corrected)=4.9, p=0.03, TT=susceptible to COPD    -   23. Genotype. MS/SS vs MM for Resistant vs COPD, Odds ratio        (OR)=2.42, 95% confidence limits 1.2-5.1, χ2 (Yates        corrected)=5.7, p=0.01, S=protective for COPD

Tissue Necrosis Factor α +489 G/A Polymorphism Allele and GenotypeFrequency in the COPD Patients and Resistant Smokers.

1. Allele* 2. Genotype Frequency A G AA AG GG COPD 54 (11%) 430 (89%) 5(2%) 44 (18%) 193 (80%) n = 242 (%) Resistant 27 (7%)  347 (93%) 1 (1%)25 (13%) 161 (86%) n = 187 (%) *number of chromosomes (2n)

-   -   1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio        (OR)=1.57, 95% confidence limits 0.9-2.7, χ² (Yates        corrected)=2.52, p=0.11,        -   AA/AG=susceptible (GG=protective)    -   2. Allele. A vs G for COPD vs resistant, Odds ratio (OR)=1.61,        95% confidence limits 1.0-2.7, χ² (Yates corrected)=3.38,        p=0.07,        -   A=susceptible

Tissue Necrosis Factor α −308 G/A Polymorphism Allele and GenotypeFrequency in the COPD Patients and Resistant Smokers.

3. Allele* 4. Genotype Frequency A G AA AG GG COPD 90 (19%) 394 (81%) 6(2%) 78 (32%) 158 (65%) n = 242 (%) Resistant 58 (15%) 322 (85%) 3 (2%)52 (27%) 135 (71%) n = 190 (%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs AG/AA for COPD vs resistant, Odds ratio        (OR)=0.77, 95% confidence limits 0.5-1.2, χ² (Yates        uncorrected)=1.62, p=0.20,        -   GG=protective (AA/AG=susceptible) trend    -   2. Allele. A vs G for COPD vs resistant, Odds ratio (OR)=1.3,        95% confidence limits 0.9-1.9, χ² (Yates uncorrected)=1.7,        p=0.20,        -   A=susceptible trend

SMAD3 C89Y Polymorphism Allele and Genotype Frequency in the COPDPatients and Resistant Smokers.

5. Allele* 6. Genotype Frequency A G AA AG GG COPD n = 250 (%) 2 (1%)498 (99%) 0 (0%) 2 (1%) 248 (99%) Resistant n = 196 6 (2%) 386 (98%) 0(0%) 6 (3%) 190 (97%) (%) *number of chromosomes (2n)

-   -   1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio        (OR)=0.26, 95% confidence limits 0.04-1.4, χ² (Yates        uncorrected)=3.19, p=0.07,        -   AA/AG=protective (GG susceptible)            Intracellular Adhesion Molecule 1 (ICAM1) A/G E469K (rs5498)            Polymorphism Allele and Genotype Frequency in COPD Patients            and Resistant Smokers.

7. Allele* 8. Genotype Frequency A G AA AG GG COPD 259 (54%) 225 (46%)73 (30%) 113 (47%) 56 (23%) n = 242 (%) Resistant 217 (60%) 147 (40%) 64(35%)  89 (49%) 29 (16%) n = 182 (%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs AG/GG for COPD vs resistant, Odds ratio        (OR)=1.60, 95% confidence limits 0.9-2.7, χ² (Yates        corrected)=3.37, p=0.07,        -   GG=susceptibility    -   2. Allele. G vs A for COPD vs resistant, Odds ratio (OR)=1.3,        95% confidence limits 1.0-1.7, χ² (Yates corrected)=2.90, p=0.09

Caspase (NOD2) Gly881Arg Polymorphism Allele and Genotype Frequencies inthe COPD Patients and Resistant Smokers.

9. Allele* 10. Genotype Frequency G C GG GC CC COPD n = 247 486 (98%)  8 (2%) 239 (97%) 8 (3%) 0 (0%) Resistant n = 195 388 (99.5%) 2 0.5%) 193(99%) 2 (1%) 0 (0%) (%) *number of chromosomes (2n)

-   -   1. Genotype. CC/CG vs GG for COPD vs resistant, Odds ratio        (OR)=3.2, 95% confidence limits 0.6-22, χ² (Yates        uncorrected)=2.41, p=0.11 (1-tailed),        -   GC/CC=susceptibility (trend)

Mannose Binding Lectin 2(MBL2) +161 G/A Polymorphism Allele and GenotypeFrequencies in the COPD Patients and Resistant Smokers.

11. Allele* 12. Genotype Frequency A G AA AG GG COPD n = 218 110 (25%)326 (75%) 6 (3%) 98 (45%) 114 (52%) (%) Resistant  66 (18%) 300 (82%) 6(3%) 54 (30%) 123 (67%) n = 183 (%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs rest for COPD vs resistant, Odds ratio        (OR)=0.53, 95% confidence limits 0.4-0.80, χ² (Yates        uncorrected)=8.55, p=0.003,        -   GG=protective

Chymase 1 (CMA1)-1903 G/A Promoter Polymorphism Allele and GenotypeFrequencies in the COPD Patients and Resistant Smokers.

13. Allele* 14. Genotype Frequency A G AA AG GG COPD 259 (54%) 219 (46%)67 (28%) 125 (52%) 47 (20%) n = 239 (%) Resistant 209 (58%) 153 (42%) 63(35%)  83 (46%) 35 (19%) n = 181 (%) *number of chromosomes (2n)

-   -   1. Genotype. AA vs AG/GG for COPD vs resistant, Odds ratio        (OR)=0.73, 95% confidence limits 0.5-1.1, χ² (Yates        corrected)=1.91, p=0.17,        -   AA genotype=protective trend

N-Acetyltransferase 2 Arg 197 Gln G/A Polymorphism Allele and GenotypeFrequencies in COPD and Resistant Smokers.

15. Allele* 16. Genotype Frequency A G AA AG GG COPD 136 (28%) 358 (72%)14 (6%)  108 (44%) 125 (50%) n = 247 (%) Resistant 125 (32%) 267 (68%)21 (11%)  83 (42%)  92 (47%) n = 196 (%) *number of chromosomes (2n)

-   -   1. Genotype. AA vs AG/GG for COPD vs resistant, Odds ratio        (OR)=0.50, 95% confidence limits 0.2-1.0, χ² (Yates        uncorrected)=3.82, p=0.05,        -   AA genotype=protective

Interleukin 1B (IL-1B) −511 A/G Polymorphism Allele and GenotypeFrequencies in COPD and Resistant Smokers.

17. Allele* 18. Genotype Frequency A G AA AG GG COPD 160 (32%) 336 (68%)31 (13%) 98 (40%) 119 (48%) n = 248 (%) Resistant 142 (36%) 248 (64%) 27(14%) 88 (45%)  80 (41%) n = 195 (%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs AA/AG for COPD vs resistant, Odds ratio        (OR)=1.3, 95% confidence limits 0.9-2.0, χ² (Yates        corrected)=1.86, p=0.17,        -   GG genotype=susceptible trend

Microsomal Epoxide Hydrolase (MEH) Tyr 113 His T/C (Exon 3) PolymorphismAllele and Genotype Frequency in COPD and Resistant Smokers.

19. Allele* 20. Genotype Frequency C T CC CT TT COPD 137 (28%) 361 (72%)18 (7%)  101 (41%) 130 (52%) n = 249 (%) Resistant 130 (34%) 258 (66%)19 (10%)  92 (47%)  83 (43%) n = 194 (%) *number of chromosomes (2n)

-   -   1. Genotype. TT vs CT/CC for COPD vs resistant, Odds ratio        (OR)=1.5, 95% confidence limits 1.0-2.2, χ² (Yates        corrected)=3.51, p=0.06,        -   TT genotype=susceptible

Microsomal Epoxide Hydrolase (MEH) His 139 Arg A/G (Exon 4) PolymorphismAllele and Genotype Frequency in COPD and Resistant Smokers.

21. Allele* 22. Genotype Frequency A G AA AG GG COPD n = 238 372 (78%)104 (22%) 148 (62%) 76 (32%) 14 (6%) (%) Resistant 277 (77%)  81 (23%)114 (64%) 49 (27%) 16 (9%) n = 179 (%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs AA/AG for COPD vs resistant, Odds ratio        (OR)=0.64, 95% confidence limits 0.3-1.4, χ² (Yates        uncorrected)=1.43, p=0.23,        -   GG genotype=protective (trend)

Lipo-Oxygenase −366 G/A Polymorphism Allele and Genotype Frequencies inthe COPD Patients and Resistant Smokers.

23. Allele* 24. Genotype Frequency A G AA AG GG COPD n = 247 21 (4%) 473(96%) 1 (0.5%) 19 (7.5%) 227 (92%) (%) Resistant 25 (7%) 359 (93%) 0(0%)   25 (13%)  167 (87%) n = 192 (%) *number of chromosomes (2n)

-   -   1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio        (OR)=0.60, 95% confidence limits 0.3-1.1, χ² (Yates        corrected)=2.34, p=0.12,        -   AA/AG genotype=protective (GG susceptible) trend

Heat Shock Protein 70 (HSP 70) HOM T2437C Polymorphism Allele andGenotype Frequencies in the COPD Patients and Resistant Smokers.

25. Allele* 26. Genotype Frequency C T CC CT TT COPD n = 199 (%) 127 2715 (3%) 117 (59%) 77 (39%) (32%) (68%) Resistant n = 166  78 254 4 (2%) 70 (42%) 92 (56%) (%) (23%) (77%) *number of chromosomes (2n)

-   -   1. Genotype. CC/CT vs TT for COPD vs resistant, Odds ratio        (OR)=2.0, 95% confidence limits 1.3-3.1, χ² (Yates        uncorrected)=9.52, p=0.002,        -   CC/CT genotype=susceptible (TT=protective)

Chloride Channel Calcium-Activated 1 (CLCA1) +13924 T/A PolymorphismAllele and Genotype Frequencies in the COPD Patients and ResistantSmokers.

27. Allele* 28. Genotype Frequency A T AA AT TT COPD n = 224 282 166 84(38%) 114 (51%) 26 (12%) (%) (63%) (37%) Resistant n = 158 178 138 42(27%)  94 (59%) 22 (14%) (%) (56%) (44%) *number of chromosomes (2n)

-   -   1. Genotype. AA vs AT/TT for COPD vs resistant, Odds ratio        (OR)=1.7, 95% confidence limits 1.0-2.7, χ² (Yates        corrected)=4.51, p=0.03,        -   AA=susceptible

Monocyte Differentiation Antigen CD-14 −159 Promoter Polymorphism Alleleand Genotype Frequencies in the COPD Patients and Resistant Smokers.

29. Allele* 30. Genotype Frequency C T CC CT TT COPD n = 240 268 212 77(32%) 114 (48%) 49 (20%) (%) (56%) (44%) Resistant n = 180 182 178 46(25%)  90 (50%) 44 (24%) (%) (51%) (49%) *number of chromosomes (2n)

-   -   1. Genotype CC vs CT/TT for COPD vs Resistant, Odds ratio        (OR)=1.4, 95% confidence limits 0.9-2.2, χ₂ (Yates        uncorrected)=2.12, p=0.15,        -   CC=susceptible (trend)

Elafin +49 C/T Polymorphism Allele and Genotype Frequencies in the COPDPatients, Resistant Smokers and Controls.

31. Allele* 32. Genotype Frequency C T CC CT TT COPD n = 144 (%) 247 41105 (73%) 37 (26%) 2 (1%) (86%) (14%) Resistant n = 75 121 29  49 (65%)23 (31%) 3 (4%) (%) (81%) (19%) *number of chromosomes (2n)

-   -   1. Genotype. CT/TT vs CC for COPD vs resistant, Odds ratio        (OR)=0.70, 95%    -   confidence limits=0.4-1.3, χ² (Yates uncorrected)=1.36, p=0.24,        -   CT/TT genotype=protective (trend only)    -   2. Allele: T vs C for COPD vs resistant, Odds ratio (OR)=0.69,        95% confidence limits=0.4-1.2, χ² (Yates uncorrected)=1.91,        p=0.17,        -   T genotype=protective (trend only)

Beta2-Adrenoreceptor Gln 27 Glu Polymorphism Allele and GenotypeFrequency in the COPD Patients, Resistant Smokers and Controls.

33. Allele* 34. Genotype Frequency C G CC CG GG Controls 204 168 57(31%) 89 (48%) 39 (21%) n = 185 (%) (55%) (45%) COPD n = 238 268 208 67(28%) 134 (56%)  37 (16%) (%) (56%) (44%) Resistant 220 170 64 (33%) 92(47%) 39 (20%) n = 195 (%) (56%) (44%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs CG/CC for COPD vs resistant, Odds ratio        (OR)=0.74, 95% confidence limits=0.4-1.2, χ² (Yates        uncorrected)=1.47, p=0.23,        -   GG=protective (trend)    -   2. Genotype. GG vs CG/CC for COPD vs controls, Odds ratio        (OR)=0.69, 95% confidence limits=0.4-1.2, χ² (Yates        uncorrected)=2.16, p=0.14,        -   GG=protective (trend)

Maxtrix Metalloproteinase 1 (MMP1) −1607 1G/2G Polymorphism Allele andGenotype Frequencies in COPD Patients, Resistant Smokers and Controls.

35. Allele* 36. Genotype Frequency 1G 2G 1G1G 1G2G 2G2G Controls n = 174214 134 68 (39%) 78 (45%) 28 (16%) (%) (61%) (39%) COPD n = 217 182 25247 (22%) 88 (41%) 82 (38%) (%) (42%) (58%) Resistant n = 187 186 188 46(25%) 94 (50%) 47 (25%) (%) (50%) (50%) *number of chromosomes (2n)

-   1. Genotype. 1G1G vs rest for COPD vs controls, Odds ratio    (OR)=0.43, 95% confidence limits 0.3-0.7, ?² (Yates    uncorrected)=13.3, p=0.0003    -   1G1G genotype=protective-   2. Allele. 1G vs 2G for COPD vs controls, Odds ration (OR)=0.45, 95%    confidence limits 0.3-0.6, ?2 (Yates corrected)=28.8, p<0.0001,    -   1G=protective-   3. Genotype. 1G1G/1G2G vs rest for COPD vs resistant smokers, Odds    ratio (OR)=0.55, 95% confidence limits 0.4-0.9, ?² (Yates    uncorrected)=6.83, p=0.009    -   1G1G/162G genotypes=protective-   4. Allele. 1G vs 2G for COPD vs resistant smokers, Odds ratio    (OR)=0.73, 95% confidence limits 0.6-1.0, ?2 (Yates corrected)=4.61,    p=0.03,    -   1G=protective-   5. Genotype. 2G2G vs 1G1G/1G2G for COPD vs controls, Odds ratio    (OR)=3.17, 95% confidence limits 1.9-5.3, ?2 (Yates    uncorrected)=21.4, p<0.0001    -   2G2G genotype=susceptible-   6. Allele. 2G vs 1G for COPD vs controls, Odds ratio (OR)=2.2, 95%    confidence limits 1.6-3.0, ?2 (Yates corrected)=28.8, p<0.00001,    -   2G=susceptible-   7. Genotype. 2G2G vs 1G1G/1G2G for COPD vs resistant, Odds ratio    (OR)=1.81, 95% confidence limits 1.2-2.9, ?2 (Yates    uncorrected)=6.83, p=0.009    -   2G2G genotype=susceptible-   8. Allele. 2G vs 1G for COPD vs resistant, Odds ratio (OR)=1.4, 95%    confidence limits 1.0-1.8, ?2 (Yates corrected)=4.61, p=0.0.03,    -   2G=susceptible

Table 2 below provides a summary of the protective and susceptibilitypolymorphisms determined for COPD.

TABLE 2 Summary of protective and susceptibility polymorphisms for COPDGene Polymorphism Role Cyclo-oxygenase 2 (COX2) COX2 −765 G/C CC/CGprotective β2-adrenoreceptor (ADBR) ADBR Arg16Gly GG susceptibleInterleukin-18 (IL18) IL18 −133 C/G CC susceptible Interleukin-18 (IL18)IL18 105 A/C AA susceptible Plasminogen activator inhibitor 1 (PAI-1)PAI-1 −675 4G/5G 5G5G susceptible Nitric Oxide synthase 3 (NOS3) NOS3298 Asp/Glu TT protective Vitamin D Binding Protein (VDBP) VDBP Lys 420Thr AA/AC protective Vitamin D Binding Protein (VDBP) VDBP Glu 416 AspTT/TG protective Glutathione S Transferase (GSTP-1) GSTP1 Ile105Val AAprotective Interferon ? (IFN-?) IFN-? 874 A/T AA susceptibleInterleukin-13 (IL13) IL13 Arg 130 Gln AA protective Interleukin-13(IL13) Il13 −1055C/T TT susceptible a1-antitrypsin (a1-AT) a1-AT Sallele MS protective Tissue Necrosis Factor α TNFa TNFa +489 G/A AA/AGsusceptible GG protective Tissue Necrosis Factor α TNFa TNFa −308 G/A GGprotective AA/AG susceptible SMAD3 SMAD3 C89Y AG AA/AG protective GGsusceptible Intracellular adhesion molecule 1 ICAM1 E469K GG susceptible(ICAM1) A/G Caspase (NOD2) NOD2 Gly 881Arg GC/CC susceptible G/C Mannosebinding lectin 2 (MBL2) MBL2 161 G/A GG protective Chymase 1 (CMA1) CMA1−1903 G/A AA protective N-Acetyl transferase 2 (NAT2) NAT2 Arg 197 GlnAA protective G/A Interleukin 1B (IL1B) (IL1B) −511 A/G GG susceptibleMicrosomal epoxide hydrolase (MEH) MEH Tyr 113 His TT susceptible T/CMicrosomal epoxide hydrolase (MEH) MEH His 139 Arg GG protective G/A 5Lipo-oxygenase (ALOX5) ALOX5 −366 G/A AA/AG protective GG susceptibleHeat Shock Protein 70 (HSP 70) HSP 70 HOM CC/CT susceptible T2437C TTprotective Chloride Channel Calcium-activated 1 CLCA1 +13924 AAsusceptible (CLCA1) T/A Monocyte differentiation antigen CD-14 CD-14−159 C/T CC susceptible Elafin Elafin Exon 1 +49 CT/TT protective C/TB2-adrenergic receptor (ADBR) ADBR Gln 27 Glu GG protective C/G Matrixmetalloproteinase 1 (MMP1) MMP1 −1607 1G1G/1G2G 1G/2G protective

The combined frequencies of the presence or absence of the selectedprotective genotypes COX2 (−765) CC/CG, β2 adreno-receptor AA,Interleukin-13 AA, Nitic Oxide Synthase 3 TT, and Vitamin D BindingProtein AA observed in the COPD subjects and in resistant smokers ispresented below in Table 3.

TABLE 3 Combined frequencies of the presence or absence of selectedprotective genotypes in COPD subjects and in resistant smokers. Numberof protective polymorphisms Cohorts 0 1 =2 Total COPD 136 (54%) 100(40%) 16 (7%)  252 Resistant smokers  79 (40%)  83 (42%) 34 (17%) 196 %of smokers with COPD 136/215 100/183 16/50 (63%) (55%) (32%) ComparisonOdd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, Resist vs COPD — — 16.430.0003 2+ vs 0-1, Resist vs COPD 3.1 1.6-6.1 12.36 0.0004 1+ vs 0,Resist vs COPD 1.74 1.2-2.6 7.71 0.006

The combined frequencies of the presence or absence of the selectedsusceptibility genotypes Interleukin-18 105 AA, PAI-1 −675 5G5G,Interleukin-13 −1055 TT, and Interferon-? −874 AA observed in the COPDsubjects and in resistant smokers is presented below in Table 4.

TABLE 4 Combined frequencies of the presence or absence of selectedsusceptibility genotypes in the COPD subjects and in resistant smokers.Number of protective polymorphisms Cohorts 0 1 =2 Total COPD 66 (26%)113 (45%) 73 (29%) 252 Resistant smokers 69 (35%)  92 (47%) 35 (18%) 196% of smokers with 66/135 113/205 73/108 COPD (49%) (55%) (68%)Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, COPD vs Resist —— 8.72 0.01 2+ vs 0-1, COPD vs Resist 1.9 1.2-3.0 6.84 0.009 1+ vs 0,COPD vs Resist 1.5 1.0-3.5 3.84 0.05

The combined frequencies of the presence or absence of the protectivegenotypes COX2 (−765) CC/CG, Interleukin-13 AA, Nitic Oxide Synthase 3TT, Vitamin D Binding Protein AA/AC, GSTP1 AA, and a1-antitypsin MS/SS,observed in the COPD subjects and in resistant smokers is presentedbelow in Table 5 and in FIG. 1.

TABLE 5 Combined frequencies of the presence or absence of selectedprotective genotypes in the COPD subjects and in resistant smokers.Number of protective polymorphisms Cohorts 0 1 =2 Total COPD 51 (19%) 64(24%) 150 (57%) 265 Resistant smokers 16 (8%)  56 (27%) 133 (65%) 205 %of smokers with COPD 51/76 64/120 150/283 (76%) (53%) (53%) ComparisonOdd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, Resist vs COPD — — 12.140.0005 1+ vs 0, Resist vs COPD 2.82 1.5-5.3 11.46 0.0004

Protective polymorphisms were assigned a score of +1 whilesusceptibility polymorphisms were assigned a score of −1. For eachsubject, a net score was then calculated according to the presence ofsusceptibility and protective genotypes. This produced a linear spreadof values. When assessed as a range between −3 to +3, a linearrelationship as depicted in FIG. 2 was observed. This analysis indicatesthat for subjects with a net score of −2 or less, there was a 70% orgreater risk of having COPD. In contrast, for subjects with a net scoreof 2+ or greater the risk was approximately 40% (see FIG. 2).

In an analysis in which the value of a given polymorphism was weightedbased on the Odd's ratio for that polymorphism (generated by comparingits frequency between resistant and COPD subjects), a linearrelationship was again observed. This analysis allowed for thedistinction of smokers at high or low risk of having COPD.

I. Example 2 Case Association Study—OCOPD Methods Subject Recruitment

Subjects of European decent who had been exposed to chronic smoking(minimum 15 pack years) and aero-pollutants in the work place (noxiousdusts or fumes) were identified from respiratory clinics. Afterspirometric testing those with occupational chronic obstructivepulmonary disease (OCOPD) with forced expiratory volume in one second(FEV1) as a percentage of predicted <70% and a FEV1/FVC ratio (Forcedexpiratory volume in one second/Forced vital capacity) of <79% (measuredusing American Thoracic Society criteria) were recruited. One hundredand thirty-nine subjects were recruited, of these 70% were male, themean FEV1/FVC (±Standard Deviation) was 54% (SD 15), mean FEV1 as apercentage of predicted was 46 (SD 19). Mean age, cigarettes per day,and pack year history was 62 yrs (SD 9), 25 cigarettes/day (SD 16) and53 pack years (SD 31), respectively. One hundred and twelve Europeansubjects who had smoked a minimum of fifteen pack years and similarlybeen exposed in the work place to potentially noxious dusts or fumeswere also studied. This control group was recruited through communitystudies of lung function and were 81% male; the mean FEV1/FVC (SD) was81% (SD 8), and mean FEV1 as a percentage of predicted was 96 (SD 10).Mean age, cigarettes per day and pack year history was 58 yrs (SD 11),26 cigarettes/day (SD 14) and 45 pack years (SD 28), respectively. Usinga PCR based method [1], all subjects were genotyped for theα1-antitrypsin mutations (M, S and Z alleles) and those with the ZZallele were excluded. The OCOPD and resistant smoker cohorts werematched for subjects with the MZ genotype (6% in each cohort). They werealso matched for age started smoking (mean 16 yr) and aged stoppedsmoking (mid fifties). 190 European blood donors (smoking andoccupational exposure status unknown) were recruited consecutivelythrough local blood donor services. Sixty-three percent were men andtheir mean age was 50 years. On regression analysis, the age differenceand pack years difference observed between OCOPD sufferers and resistantsmokers was found not to determine FEV or OCOPD.

Summary of Characteristics for the OCOPD and Exposed Resistant SmokerCohorts.

Parameter OCOPD Exposed resistant Mean (SD) (N = 139) smokers (N = 112)Differences % male 70% 81% P < 0.05 Age (yrs) 62 (9)  58 (11) ns Packyears 53 (31) 45 (28) P < 0.05 Cigarettes/day 25 (16) 26 (14) ns FEV1(L) 1.3 (0.7) 3.0 (0.7) P < 0.05 FEV1 % predict 46 (19) 96% (10)   P <0.05 FEV1/FVC 54 (15) 81 (8)  P < 0.05

Means and 1SD

Cyclooxygenase 2 (COX2) −765 G/C Promoter Polymorphism anda1-Antitrypsin Genotyping

Genomic DNA was extracted from whole blood samples [2]. The COX2 −765polymorphism was determined by minor modifications of a previouslypublished method [3]. The PCR reaction was carried out in a total volumeof 25 ul and contained 20 ng genomic DNA, 500 pmol forward and reverseprimers, 0.2 mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.0 mM MgCl₂and 1 unit of Taq polymerase (Life Technologies). Cycling times wereincubations for 3 min at 95° C. followed by 33 cycles of 50 s at 94° C.,60 s at 66° C. and 60 s at 72° C. A final elongation of 10 min at 72° C.then followed. 4 ul of PCR products were visualised by ultraviolettrans-illumination of a 6% agarose gel stained with ethidium bromide. Analiquot of 3 ul of amplification product was digested for 1 hr with 4units of AciI (Roche Diagnostics, New Zealand) at 37° C. Digestedproducts were separated on a 2.5% agarose gel run for 2.0 hrs at 80 mVwith TBE buffer and visualised using ultraviolet transillumination afterethidium bromide staining against a 123 bp ladder. Using a PCR basedmethod discussed above [3], all smoking subjects were genotyped for theα1-antitrypsin M, S and Z alleles.

Genotyping of the Superoxide Dismutase 3 Arg 312 Gln Polymorphism

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described [4,herein incorporated by reference in its entirety]. Genotyping was doneusing minor modifications of the above protocol optimised for laboratoryconditions. The PCR reactions were amplified in MJ Researchthermocyclers in a total volume of 25 μl and contained 80 ng genomicDNA, 10 pmol forward and reverse primers, 0.1 mM dNTPs, 10 mM Tris-HCL(pH 8.4), 150 mM KCl, 1.0 mM MgCl₂ and 0.5 unit of Taq polymerase(Qiagen). Aliquots of amplification product were digested for 4 hrs with5 U of the relevant restriction enzymes (Roche Diagnostics, New Zealand)at designated temperatures and conditions. Digested products wereseparated on 8% polyacrylamide gels (49:1, Sigma). The products werevisualised by ultraviolet transillumination following ethidium bromidestaining and migration compared against a 1 Kb plus ladder standard(Invitrogen). Genotypes were recorded in data spreadsheets andstatistical analysis performed.

Genotyping of the Microsomal Epoxide Hydrolase Exon 3 TC Polymorphism

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described [5,herein incorporated by reference in its entirety]. Genotyping was doneusing minor modifications of the above protocol optimised for laboratoryconditions. The PCR reactions were amplified in MJ Researchthermocyclers in a total volume of 25 μl and contained 80 ng genomicDNA, 100 ng forward and reverse primers, 0.2 mM dNTPs, 10 mM Tris-HCL(pH 8.4), 150 mM KCl, 1.5 mM MgCl₂ and 1.0 unit of Taq polymerase(Qiagen). Cycling conditions consisted of 94° C. 60 s, 56° C. 20 s, 72°C. 20 s for 38 cycles with an extended last extension of 3 min. Aliquotsof amplification product were digested for 4 hrs with 5 U of therelevant restriction enzymes Eco RV (Roche Diagnostics, New Zealand) atdesignated temperature conditions. Digested products were separated on8% polyacrylamide gels (49:1, Sigma). The products were visualised byultraviolet transillumination following ethidium bromide staining andmigration compared against a 1 Kb plus ladder standard (Invitrogen).Genotypes were recorded in data spreadsheets and statistical analysisperformed.

Genotyping of the 3′ 1237 G/A (T/T) Polymorphism of the a1-AntitrypsinGene

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described[Sandford A J et al., [6], each of which is herein incorporated byreference in its entirety]. Genotyping was done using minormodifications of the above protocol optimised for laboratory conditionsThe PCR reactions were amplified in MJ Research thermocyclers in a totalvolume of 25 μl and contained 80 ng genomic DNA, 100 ng forward andreverse primers, 0.2 mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.5mM MgCl₂ and 1.0 unit of Taq polymerase (Qiagen). Forward and reverseprime sequences were 5′-CTACCAGGAATGGCCTTGTCC-3′ [SEQ.ID.NO.136] and5′-CTCTCAGGTCTGGTGTCATCC-3′ [SEQ.ID.NO.137]. Cycling conditionsconsisted of 94 C 60 s, 56 C 20 s, 72 C 20 s for 38 cycles with anextended last extension of 3 min. Aliquots of amplification product weredigested for 4 hrs with 2 Units of the restriction enzymes Taq 1 (RocheDiagnostics, New Zealand) at designated temperature conditions. Digestedproducts were separated on 3% agarose. The products were visualised byultraviolet transillumination following ethidium bromide staining andmigration compared against a 1 Kb plus ladder standard (Invitrogen).Genotypes were recorded in data spreadsheets and statistical analysisperformed.

Genotyping of the Asp 299 Gly Polymorphism of the Toll-Like Receptor 4Gene

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described [6].Genotyping was done using minor modifications of the above protocoloptimised for laboratory conditions The PCR reactions were amplified inMJ Research thermocyclers in a total volume of 25 μl and contained 80 nggenomic DNA, 100 ng forward and reverse primers, 0.2 mM dNTPs, 10 mMTris-HCL (pH 8.4), 150 mM KCl, 1.5 mM MgCl₂ and 1.0 unit of Taqpolymerase (Qiagen). Forward and reverse prime sequences were5′-GATTAGCATACTTAGACTACTACCTCCATG-3′ [SEQ.ID.NO.138] and5′-GATCAACTTCTGAAAAAGCATTCCCAC-3′ [SEQ.ID.NO.139]. Cycling conditionsconsisted of 94° C. 30 s, 55° C. 30 s, 72° C. 30 s for 30 cycles with anextended last extension of 3 min. Aliquots of amplification product weredigested for 4 hrs with 2 U of the restriction enzyme Nco I (RocheDiagnostics, New Zealand) at designated temperature conditions. Digestedproducts were separated on 3% agarose gel. The products were visualisedby ultraviolet transillumination following ethidium bromide staining andmigration compared against a 1 Kb plus ladder standard (Invitrogen).Genotypes were recorded in data spreadsheets and statistical analysisperformed.

Genotyping of the −1607 1G2G Polymorphism of the MatrixMetalloproteinase 1 Gene

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described[Dunleavey L, et al]. Genotyping was done using minor modifications ofthe above protocol optimised for laboratory conditions The PCR reactionswere amplified in MJ Research thermocyclers in a total volume of 25 μland contained 80 ng genomic DNA, 100 ng forward and reverse primers, 200mM dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂ and 1.0 unitof Taq polymerase (Qiagen). Forward and reverse prime sequences were 3′TCGTGAGAATGTCTTCCCATT-3′ [SEQ.ID.NO.140] and5′-TCTTGGATTGATTTGAGATAAGTGAAATC-3′ [SEQ.ID.NO.141]. Cycling conditionsconsisted of 94 C 60 s, 55 C 30 s, 72 C 30 s for 35 cycles with anextended last extension of 3 min. Aliquots of amplification product weredigested for 4 hrs with 6 Units of the restriction enzymes XmnI (RocheDiagnostics, New Zealand) at designated temperature conditions. Digestedproducts were separated on 6% polyacrylamide gel. The products werevisualised by ultraviolet transillumination following ethidium bromidestaining and migration compared against a 1 Kb plus ladder standard(Invitrogen). Genotypes were recorded in data spreadsheets andstatistical analysis performed.

Other Polymorphism Genotyping

Genomic DNA was extracted from whole blood samples [4]. Purified genomicDNA was aliquoted (10 ng/ul concentration) into 96 well plates andgenotyped on a Sequenom™ system (Sequenom™ Autoflex Mass Spectrometerand Samsung 24 pin nanodispenser) using the sequences, amplificationconditions and methods described below.

The following conditions were used for the PCR multiplex reaction: finalconcentrations were for 10× Buffer 15 mM MgCl2 1.25×, 25 mM MgCl2 1.625mM, dNTP mix 25 mM 500 uM, primers 4 uM 100 nM, Taq polymerase (Quiagenhot start) 0.15 u/reaction, Genomic DNA 10 ng/ul. Cycling times were 95°C. for 15 min, (5° C. for 15 s, 56° C. 30 s, 72° C. 30 s for 45 cycleswith a prolonged extension time of 3 min to finish. We used shrimpalkaline phosphotase (SAP) treatment (2 ul to 5 ul PCR reaction)incubated at 35° C. for 30 min and extension reaction (add 2 ul to 7 ulafter SAP treatment) with the following volumes per reaction of water0.76 ul, hME 10× termination buffer 0.2 ul, hME primer (10 uM) 1 ul,MassEXTEND enzyme 0.04 ul.

Sequenom Conditions for the Polymorphisms Genotyping—1

SNP_ID TERM WELL 2nd-PCRP 1st-PCRP VDBP - 420 ACT W1ACGTTGGATGGCTTGTTAACCAGCTT ACGTTGGATGTTTTTCAGACTGGCAG TGCC [SEQ. ID. NO.142] AGCG [SEQ. ID. NO. 143] VDBP - 416 ACT W1ACGTTGGATGTTTTTCAGACTGGCAG ACGTTGGATGGCTTGTTAACCAGCTTT AGCG [SEQ. ID.NO. 144] GCC [SEQ. ID. NO. 145] ADRB2- ACT W2 ACGTTGGATGTTGCTGGCACCCAATGACGTTGGATGATGAGAGACATGACGA Gln27Glu GAAG [SEQ. ID. NO. 146] TGCC [SEQ.ID. NO. 147] GSTP1 -105 ACT W2 ACGTTGGATGTGGTGGACATGGTGAAACGTTGGATGTGGTGCAGATGCTCAC TGAC [SEQ. ID. NO. 148] ATAG [SEQ. ID. NO.149] PAI1 G-675G ACT W2 ACGTTGGATGCACAGAGAGAGTCTGGACGTTGGATGCTCTTGGTCTTTCCCTC ACAC [SEQ. ID. NO. 150] ATC [SEQ. ID. NO.151] IL-11 G518A ACT W3 ACGTTGGATGCCTCTGATCCTCTTTGCACGTTGGATGAAGAGGGAGTGGAAG TTC [SEQ. ID. NO. 152] GGAAG [SEQ. ID. NO.153] NOS3 - 298 ACT W3 ACGTTGGATGACAGCTCTGCATTCAGACGTTGGATGAGTCAATCCCTTTGGT CACG [SEQ. ID. NO. 154] GCTC [SEQ. ID. NO.155] IL-8 A-251T CGT W5 ACGTTGGATGACTGAAGCTCCACAATACGTTGGATGGCCACTCTAGTACTAT TTGG [SEQ. ID. NO. 156] ATCTG [SEQ. ID. NO.157] IL-18 C-133G ACT W6 ACGTTGGATGGGGTATTCATAAGCTGACGTTGGATGCCTTCAAGTTCAGTGG AAAC [SEQ. ID. NO. 158] TCAG [SEQ. ID. NO.159] IL-18 A105C ACT W8 ACGTTGGATGGGTCAATGAAGAGAAACGTTGGATGAATGTTTATTGTAGAA CTTGG [SEQ. ID. NO. 160] AACC [SEQ. ID. NO.161]

Sequenom Conditions for the Polymorphisms Genotyping—2

SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC PWARN UEP_DIR VDBP - 420 9999.7 99.7 46.2 53.3 ML R VDBP - 416 99 99.7 99.7 45.5 33.3 M FADRB2-Gln27Glu 118 96.6 80 52.2 66.7 L F GSTP1 -105 107 99.4 80 49.952.9 F PAI1 G-675G 109 97.9 80 59.3 66.7 g F IL-11 G518A 169 97.5 6552.9 52.6 s F NOS3 - 298 186 98.1 65 61.2 63.2 F IL-8 A-251T 119 92.681.2 45.9 28.6 R IL-18 C-133G 112 93.5 74.3 41.8 46.7 L F IL-18 A105C121 67.2 74.3 48.9 40 R

Sequenom Conditions for the Polymorphisms Genotyping—3

SNP_ID UEP_MASS UEP_SEQ EXT1_CALL EXT1_MASS VDBP - 420 4518.9AGCTTTGCCAGTTCC[SEQ. ID. NO. 162] A 4807.1 VDBP - 416 5524.6AAAAGCAAAATTGCCTGA[SEQ. ID. NO. T 5812.8 163] ADRB2- 4547CACGACGTCACGCAG[SEQ. ID. NO. 164] C 4820.2 Gln27Glu GSTP1 -105 5099.3ACCTCCGCTGCAAATAC[SEQ. ID. NO. 165] A 5396.5 PAI1 G-675G 5620.6GAGTCTGGACACGTGGGG[SEQ. ID. NO. DEL 5917.9 166] IL-11 G518A 5705.7TCCATCTCTGTGGATCTCC[SEQ. ID. NO. A 6002.9 167] NOS3 - 298 5813.8TGCTGCAGGCCCCAGATGA[SEQ. ID. NO. T 6102 168] IL-8 A-251T 6428.2CACAATTTGGTGAATTATCAA[SEQ. ID. A 6716.4 NO. 169] IL-18 C-133G 4592AGCTGAAACTTCTGG[SEQ. ID. NO. 170] C 4865.2 IL-18 A105C 6085TCAAGCTTGCCAAAGTAATC[SEQ. ID. A 6373.2 NO. 171]

Sequenom Conditions for the Polymorphisms Genotyping—4

SNP_ID EXT1_SEQ EXT2_CALL EXT2_MASS EXT2_SEQ 1stPAUSE VDBP - 420AGCTTTGCCAGTTCCT C 5136.4 AGCTTTGCCAGTTCCGT 4848.2 [SEQ. ID. NO. 172][SEQ. ID. NO. 173] VDBP - 416 AAAAGCAAAATTGCCTGA G 6456.2AAAAGCAAAATTGCCTGAG 5853.9 T [SEQ. ID. NO. 174] GC [SEQ. ID. NO. 175]ADRB2- CACGACGTCACGCAGC G 5173.4 CACGACGTCACGCAGGA 4876.2 Gln27Glu [SEQ.ID. NO. 176] [SEQ. ID. NO. 177] GSTP1 -105 ACCTCCGCTGCAAATACA G 5716.7ACCTCCGCTGCAAATACGT 5428.5 [SEQ. ID. NO. 178] [SEQ. ID. NO. 179] PAI1G-675G GAGTCTGGACACGTGGGG G 6247.1 GAGTCTGGACACGTGGGGG 5949.9 A [SEQ.ID. NO. 180] A [SEQ. ID. NO. 181] IL-11 G518A TCCATCTCTGTGGATCTCC G6323.1 TCCATCTCTGTGGATCTCC 6034.9 A [SEQ. ID. NO. 182] GT [SEQ. ID. NO.183] NOS3 - 298 TGCTGCAGGCCCCAGATG G 6416.2 TGCTGCAGGCCCCAGATGA 6143 AT[SEQ. ID. NO. 184] GC [SEQ. ID. NO. 185] IL-8 A-251T CACAATTTGGTGAATTATT 7029.6 CACAATTTGGTGAATTATC 6741.4 CAAT [SEQ. ID. NO. 186] AAAT [SEQ.ID. NO. 187] IL-18 C-133G AGCTGAAACTTCTGGC G 5218.4 AGCTGAAACTTCTGGGA4921.2 [SEQ. ID. NO. 188] [SEQ. ID. NO. 189] IL-18 A105CTCAAGCTTGCCAAAGTAA C 7040.6 TCAAGCTTGCCAAAGTAAT 6414.2 TCT [SEQ. ID. NO.190] CGGA[SEQ. ID. NO. 191]

Results

Frequencies of individual polymorphisms are as follows:

TABLE 6 Polymorphism allele and genotype frequency in the OCOPDpatients, exposed resistant smokers and controls. Cyclo-oxygenase 2 −765G/C Allele* Genotype Frequency C G CC CG GG Controls n = 95 (%) 27 (14%)161 (86%) 3 (3%) 21 (22%) 70 (75%) OCOPD n = 82 (%) 22 (13%) 142⁴ (87%) 2 (2%) 18 (22%) 62³ (76%)  Resistant n = 87 (%) 42² (24%)  132 (76%) 6¹(7%)  30¹ (34%)  51 (59%) Glutathione S Transferase P1 Ile 105 Val (A/G)Allele* Genotype Frequency A G AA AG GG Controls n = 186 (%) 234 (63%)138 (37%)  71 (38%) 92 (50%) 23 (12%) OCOPD n = 123 (%) 159 (65%) 87(36%) 52 (42%) 55 (45%) 16⁵ (13%)  Resistant n = 98 (%) 136 (69%) 60(31%) 44 (45%) 48 (49%) 6 (6%) Interleukin 18 105 C/A Allele* GenotypeFrequency C A CC AC AA Controls n = 185 (%) 119 (32%)  251 (68%) 22(12%) 75 (40%)   88 (48%) OCOPD n = 122 (%) 62 (25%) 182 (75%) 12 (10%)38 (31%) 72^(6,7) (59%) Resistant n = 98 (%) 60 (31%) 136 (69%)  6 (6%)48 (49%)   44 (45%) Interleukin 18 −133 G/C Allele* Genotype Frequency GC GG GC CC Controls n = 188 (%) 121 (32%)  255 (68%) 23 (12%) 75 (40%)  90 (48%) OCOPD n = 122 62 (25%) 182 (75%) 12 (10%) 38 (31%) 72^(8,9)(59%) Resistant n = 97 (%) 60 (31%) 134 (69%) 6 (6%) 48 (50%)   43 (44%)Interleukin 8 −251 A/T Allele* Genotype Frequency A T AA AT TT Controlsn = 188 (%) 175 (47%) 201 (53%)  39 (21%) 97 (52%) 52 (28%) OCOPD n =116 101 (44%) 131 (56%)  21 (18%) 59 (51%) 36 (31%) Resistant n = 93 (%)94¹¹ (50%)   92 (49%) 26¹⁰ (28%) 42 (45%) 25 (27%) Vitamin D BindingProtein Lys 420 Thr (A/C) Allele* Genotype Frequency A C AA AC CCControls n = 189 (%)  113 (30%) 265 (70%)  17 (9%)  79 (42%)  93 (49%)OCOPD n = 122 (%)   62 (25%) 182 (75%)  5 (4%) 52 (43%) 65¹⁴ (53%)Resistant n = 99 (%)  73¹³ (37%) 125 (63%) 12¹² (12%) 49 (50%)  38 (38%)Vitamin D Binding Protein Glu 416 Asp (T/G) Allele* Genotype Frequency TG TT TG GG Controls n = 189 (%)  163 (43%) 215 (57%)  35 (19%)  93 (49%) 61 (32%) OCOPD n = 122 (%)  109 (45%) 135 (55%)  25 (21%)  59 (48%)38¹⁷ (31%) Resistant n = 99 (%) 103¹⁶ (52%)  95 (48%) 23¹⁵ (23%) 57¹⁵(58%)  19 (19%) Microsomal epxoide hydrolase R/r Exon 3 T/C Allele*Genotype Frequency r R rr Rr RR Controls n = 184 (%) 228 (62%) 140(38%)  77 (42%) 74 (40%)  33 (18%) OCOPD n = 98 (%) 144 (74%) 52 (26%)55 (56%) 34 (35%)  9 (9%) Resistant n = 102 (%) 135 (66%) 69 (34%) 52(51%) 31 (30%) 19¹⁸ (19%) Super oxide dismutase 3 Arg 312 Gln Allele*Genotype Frequency A G AA AG GG Controls n = 190 (%)  371 (98%)  9 (2%)183 (96%)   5 (3%)  2 (1%) OCOPD n = 100 (%) 199²⁰ (99%)  1 (1%) 99(99%)  1 (1%)  0 (0%) Resistant n = 102 (%)  193 (95%) 11²⁰ (5%)  92(90%) 9¹⁹ (9%) 1¹⁹ (1%) a1-antitrypsin S Allele* Genotype Frequency M SMM MS SS OCOPD n = 88 (%) 171 (97%)   5 (3%) 83 (94%)  5 (6%) 0 (0%)Resistant n = 94 (%) 175 (93%) 13²² (7%) 81 (86%) 13²¹ (14%) 0 (0%)Toll-like receptor 4 Asp 299 Gly A/G Allele* Genotype Frequency A G AAAG GG OCOPD n = 60 (%) 117 (98%) 1 (2%) 58 (98%)  1 (2%) 0 (0%)Resistant n = 34 (%)  65 (96%) 3 (4%) 31 (91%) 3²³ (9%) 0 (0%)Beta2-adrenoreceptor Gln 27 Glu Allele* Genotype Frequency C G CC CG GGControls n = 186 (%) 204 (55%) 168 (45%)  57 (31%) 90 (48%) 39 (21%)OCOPD n = 122 (%) 129 (53%) 115 (47%)  32 (26%) 65 (53%) 25 (21%)Resistant n = 99 (%) 117 (59%)  81 (41%) 38²⁴ (38%) 41 (41%) 20 (20%)Interleukin 11 (IL-11) −518 G/A Allele* Genotype Frequency A G AA AG GGOCOPD n = 119 (%) 110 (46%) 128 (54%)  22 (19%) 66 (55%) 31 (26%)Resistant n = 98 (%) 103 (53%)  93 (47%) 26²⁵ (27%) 51 (52%) 21 (21%)Interleukin-13 −1055 C/T Allele* Genotype Frequency T C TT TC CCControls n = 182 (%) 65 (18%) 299 (82%)  5 (3%) 55 (30%) 122 (67%) OCOPD n = 121 (%) 53 (22%) 189 (78%) 5²⁶ (4%) 43 (36%) 73 (60%)Resistant n = 97 (%) 31 (16%) 163 (84%)  1 (1%) 29 (30%) 67 (69%)Plasminogen activator inhibitor 1 −675 4G/5G Allele* Genotype Frequency5G 4G 5G5G 5G4G 4G4G Controls n = 186 (%)  158 (42%) 214 (58%)  31 (17%)96 (52%) 59 (32%) OCOPD n = 122 (%) 115²⁸ (47%) 129 (53%) 29²⁷ (24%) 57(47%) 36 (30%) Resistant n = 98 (%)   76 (39%) 120 (61%)  14 (14%) 48(49%) 36 (37%) Nitric oxide synthase 3 Asp 298 Glu (T/G) Allele*Genotype Frequency T G TT TG GG Controls n = 183 (%) 108 (30%)  258(70%)    13 (7%) 82 (45%) 88 (48%) OCOPD n = 120 (%) 71 (30%) 169 (70%)   10 (8%) 51 (43%) 59 (49%) Resistant n = 99 (%) 71 (36%) 127 (64%)15^(29,30) (15%) 41 (41%) 43 (43%) a1-antitrypsin 3′ 1237 G/A (T/t)Allele* Genotype Frequency T t TT Tt tt Controls n = 178 (%) 345 (97%)11 (3%)   167 (94%)  11 (6%)   0 (0%)  COPD n = 61 (%) 109 (89%) 13(11%)³² 50 (82%)  9 (15%)³¹ 2 (3%)³¹ Resistant n = 35 (%)  67 (96%) 3(4%)  32 (91%) 3 (9%)  0 (0%)  Matrix metalloproteinase 1 −1607 1G/2GAllele* Genotype Frequency 1G 2G 1G1G 1G2G 2G2G Controls n = 174 (%) 214(61%)  134 (39%)  68 (39%) 78 (45%) 28 (16%)  COPD n = 93 (%) 90 (48%) 96 (52%)³⁴ 24 (26%) 42 (45%) 27 (29%)³³ Resistant n = 94 (%) 99 (53%) 89 (47%)  25 (27%) 49 (52%) 20(21%)  *number of chromosomes (2n)

-   -   1. Genotype. CC/CG vs GG for resistant vs OCOPD, Odds ratio        (OR)=2.2, 95% confidence limits=1.1-4.8, χ² (Yates        corrected)=4.76, P=1.03, CC/CG=protective    -   2. Allele. C vs G for resistant vs OCOPD, Odds ratio (OR)=2.1,        95% confidence limits 1.1-3.8, χ² (Yates corrected)=5.65,        p=0.02. C=protective    -   3. Genotype. GG vs CG/CC for OCOPD vs resistant, Odds ratio        (OR)=0.5, 95% confidence limits=0.2-0.9, χ² (Yates        corrected)=4.76, P=0.03. GG=susceptible    -   4. Allele. G vs C for OCOPD vs resistant, Odds ratio (OR)=0.5,        95% confidence limits 0.3-0.9, χ² (Yates corrected)=5.65,        p=0.02. G=susceptible    -   5. Genotype. GG vs AG/AA for OCOPD vs resistant, Odds ratio        (OR)=2.3, 95% confidence limits=0.8-6.9, χ² (Yates        uncorrected)=2.88, p=0.09. GG genotype=susceptible    -   6. Genotype. AA vs AC/CC for OCOPD vs resistant, Odds ratio        (OR)=1.8, 95% confidence limits=1.0-3.1, χ² (Yates        corrected)=3.8, p=0.05. AA=susceptibility    -   7. Genotype. AA vs AC/CC for OCOPD vs controls, Odds ratio        (OR)=1.6, 95% confidence limits 1.0-2.6, χ² (Yates        uncorrected)=3.86, p=0.05. AA=susceptibility    -   8. Genotype. CC vs CG/GG for OCOPD vs controls, Odds ratio        (OR)=1.6, 95% confidence limits=1.0-2.6, χ² (Yates        uncorrected)=3.68, p=0.05. CC=susceptibility    -   9. Genotype. CC vs CG/GG for OCOPD vs resistant, Odds ratio        (OR)=1.8, 95% confidence limits 1.0-3.2, χ² (Yates        corrected)=4.10, p=0.04. CC=susceptibility    -   10. Genotype. AA vs AT/TT for OCOPD vs resistant, Odds ratio        (OR)=1.8, 95% confidence limits=0.9-3.6, χ² (Yates        uncorrected)=2.88, p=0.09. AA=protective    -   11. Allele. A vs T for OCOPD vs resistant, Odds ratio (OR)=1.3,        95% confidence limits=0.9-2.0, χ² (Yates uncorrected)=2.3,        p=0.15. A=protective    -   12. Genotype. AA vs AC/CC for resistant vs OCOPD, Odds ratio        (OR)=3.2, 95% confidence limits=1.0-11.0, χ² (Yates        corrected)=3.89, p=0.05. AA genotype=protective    -   13. Allele. A vs C for resistant vs OCOPD, Odds ratio (OR)=1.7,        95% confidence limits 1.1-2.6, χ² (Yates corrected)=6.24,        p=0.01. A allele=protective    -   14. Genotype. CC vs AC/AA for OCOPD vs resistant, Odds ratio        (OR)=1.8, 95% confidence limits=1.0-3.3, χ² (Yates        corrected)=4.29, p=0.04. CC genotype=susceptibility    -   15. Genotype. TT/TG vs GG for resistant vs OCOPD, Odds ratio        (OR)=1.9, 95% confidence limits=1.0-38, χ² (Yates        uncorrected)=4.08, p=0.04. TT/TG genotype=protective    -   16. Allele. T vs G for resistant vs OCOPD, Odds ratio (OR)=1.3,        95% confidence limits 0.9-2.0, χ² (Yates uncorrected)=2.36,        p=0.12. A allele=protective    -   17. Genotype. GG vs TT/TG for OCOPD vs resistant, Odds ratio        (OR)=0.5, 95% confidence limits=0.3-1.0, χ² (Yates        uncorrected)=4.1, p=0.04. GG genotype=susceptible    -   18. Genotype. RR vs Rr/rr for resistant vs OCOPD, Odds ratio        (OR)=2.3, 95% confidence limits=0.9-5.8, χ² (Yates        uncorrected)=3.7, p=0.05, RR genotype=protective    -   19. Genotype. AG/GG vs AA for resistant vs OCOPD, Odds ratio        (OR)=10.8, 95% confidence limits=1.4-229, χ² (Yates        corrected)=5.99 p=0.01. AG/GG genotype=protective, AA        susceptible    -   20. Allele. G vs A for resistant vs OCOPD, Odds ratio (OR)=11.3,        95% confidence limits 1.5-237, χ² (Yates corrected)=6.77,        p=0.001. G allele=protective, A susceptible    -   21. Genotype. MS vs MM for Resistant vs OCOPD, Odds ratio        (OR)=2.7, 95% confidence limits 0.8-9.0, χ² (Yates        uncorrected)=3.4, p=0.07.        -   MS=protective    -   22. Allele: S vs M allele for resistant vs OCOPD, Odds ratio        (OR)=2.5, 95% confidence limits 0.8-8.4, χ² (Yates        uncorrected)=3.24, p=0.07.    -   23. Genotype AG vs AA in resistant vs OCOPD, Odd's Ratio        (OR)=5.61, 95% confidence limits 0.5-146, χ² (Yates        uncorrected)=2.66, p=0.10. AG=protective    -   24. Genotype. CC vs CG/GG for resistant vs OOCOPD, Odds ratio        (OR)=1.75, 95% confidence limits=1.0-3.2, χ² (Yates        uncorrected)=3.73, p=0.05. CC=protective    -   25. Genotype: AA vs AG/GG for resistant vs OCOPD, Odd's Ratio        (OR)=1.6, 95% confidence limits 0.8-32, χ² (Yates        uncorrected)=2.02, p=0.16. AA=protective    -   26. Genotype. TT vs TC/CC for OCOPD vs resistant, Odds ratio        (OR)=6.03, 95% confidence limits 1.1-42, χ² (Yates        corrected)=4.9, p=0.03. TT=susceptible    -   27. Genotype. 5G5G vs rest for OCOPD vs resistant, Odds ratio        (OR)=1.9, 95% confidence limits 0.9-4.0, χ² (Yates        uncorrected)=3.11, p=0.08. 5G5G=susceptible    -   28. Allele. 50 vs 4G for OCOPD vs resistant, Odds ratio        (OR)=1.4, 95% confidence limits 0.9-2.1, χ² (Yates        corrected)=3.1, p=0.08. 5G=susceptible    -   29. Genotype. TT vs TG/GG for resistant vs controls, Odds ratio        (OR)=2.3, 95% confidence limits 1.0-5.5, χ² (Yates        corrected)=3.80, p=0.05. TT genotype=protective    -   30. Genotype. TT vs TG/GG for resistant vs OCOPD, Odds ratio        (OR)=1.9, 95% confidence limits 0.8-5.0, χ² (Yates        uncorrected)=2.49, p=0.11. TT genotype=protective    -   31. Genotype: Tt/tt vs TT for COPD vs controls, Odd's Ratio        (OR)=3.34, 95% confidence limits 1.3-8.9, χ² (Yates        corrected)=6.28, p=0.01. Tt/tt susceptible to OCOPD    -   32. Allele: t vs T for COPD vs controls, Odd's Ratio (OR)=2.5,        95% confidence limits 1.0-6.3, χ² (Yates corrected)=4.1, p=0.04.        t=susceptible to OCOPD    -   33. Genotype. 2G2G vs 1G1G/1G2G for COPD vs controls, Odds ratio        (OR)=2.1, 95% confidence limits 1.1-4.1, χ² (Yates        corrected)=5.44, p=0.02. 2G2G genotype=susceptible for OCOPD    -   34. Allele. 2G vs 1G for COPD vs controls, Odds ratio (OR)=1.7,        95% confidence limits 1.2-2.5, χ² (Yates corrected)=7.97,        p=0.005. 2G=susceptible for OCOPD

Table 7 below provides a summary of the protective and susceptibilitypolymorphisms determined for OCOPD.

TABLE 7 Summary of protective and susceptibility polymorphisms for OCOPDGene Polymorphism Role Cyclo-oxygenase (Cox) 2 Cox 2 −765 G/C CC/CGprotective GG susceptible β2-adrenoreceptor (ADRB2) ADRB2 Gln 27Glu CCprotective Interleukin-18 (IL-18) IL-18 −133 C/G CC susceptibleInterleukin-18 (IL-18) IL-18 105 A/C AA susceptible Plasminogenactivator PAI-1 −675 4G/5G 5G5G inhibitor 1 (PAI-1) susceptible NitricOxide synthase 3 (NOS3) NOS3 298 Asp/Glu TT protective Vitamin D BindingProtein VDBR Lys 420 Thr AA protective (VDBR) CC susceptible Vitamin DBinding Protein VDBP Glu 416 Asp TT/TG protective (VDBR) GG susceptibleGlutathione S Transferase GSTP1 Ile105Val GG susceptible (GSTP1)Superoxide dismutase 3 (SOD3) SOD3 Arg 312 Gln AG/GG protective AAsusceptible a1-antitrypsin (a1AT) a1AT 3′ 1237 G/A Tt/tt susceptible(T/t) a1-antitrypsin (a1AT) a1AT S allele MS protective Toll-likereceptor 4 (TLR4) TLR4 Asp 299 Gly AG/GG A/G protective Interleukin-8(IL-8) IL-8 −251 A/T AA protective Interleukin 11 (IL-11) IL-11 −518 G/AAA protective Microsomal epoxide MEH Exon 3 T/C RR protective hydrolase(MEH) (r/R) Interleukin 13 (IL-13) IL-13 −1055 C/T TT susceptible MatrixMetalloproteinase 1 MMP1 −1607 2G2G susceptible (MMP1) 1G/2G

The combined frequencies of the presence or absence of the selectedprotective genotypes COX2 −765 CC/CG, NOS3 298 TT, a 1AT MS/SS, SOD3AG/GG, MEH Exon 3 RR, and VDBP 420 AA observed in the OCOPD subjects andin resistant smokers is presented below in Table 8.

TABLE 8 Combined frequencies of the presence or absence of protectivegenotypes in OCOPD subjects and in resistant smokers. Number ofprotective polymorphisms Cohorts 0 1 =2 Total OCOPD 34 (27%) 51 (41%) 39(32%) 124 Resistant 20 (19%) 31 (30%) 53 (51%) 104 smokers % of 34/54(63%)   51/82 (62%)   39/92 (42%)   smokers with OCOPD Comparison Odd'sratio 95% CI ?2 P value 0 vs 1 · vs 2+, Resist vs OCOPD — — 16.2 0.0032+ vs 0-1, Resist vs OCOPD 2.3 1.3-4.0 8.15 0.004 0 vs 2+, OCOPD vsResist 2.3 1.1-4.9 4.97 0.03

The combined frequencies of the presence or absence of the selectedsusceptibility genotypes MMP1−1607 2G2G, GSTP1 105 GG, PAI-1-675 5G5G,IL-13 −1055 TT, and VDBP 416 GG, observed in the OCOPD subjects and inresistant smokers is presented below in Table 9.

TABLE 9 Combined frequencies of the presence or absence of selectedsusceptibility genotypes in OCOPD subjects and in resistant smokers.Number of protective polymorphisms Cohorts 0 1 =2 Total OCOPD 45 (36%)55 (44%) 24 (20%) 124 Resistant 55 (54%) 37 (37%) 9 (9%) 101 smokers %of 45/100 (45%)    55/92 (60%)   24/33 (73%)   smokers with OCOPDComparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, OCOPD vs Resist —— 9.1 0.01 2+ vs 0-1, OCOPD vs Resist 2.5 1.0-6.0 4.05 0.04 0+ vs 1-2+,Resist vs OCOPD 2.1 1.2-3.7 6.72 0.01

Protective polymorphisms were assigned a score of +1 whilesusceptibility polymorphisms were assigned a score of −1. For eachsubject, a net score was then calculated according to the presence ofsusceptibility and protective genotypes. This produced a linear spreadof values, as shown in Table 10. When assessed as a range between −2 to+3, a linear relationship as depicted in FIG. 3 was observed. Thisanalysis indicates that for subjects with a net score of −1 or less,there was an approximately 70% or greater risk of having OCOPD. Incontrast, for subjects with a net score of 2+ or greater, the risk wasapproximately 25% (see FIG. 3). As a point of clarification, it is notedthat FIG. 3 depicts the sum of the protective and susceptibilitypolymorphisms combined, rather than simply the sum of the protectivepolymorphisms in one graph and the sum of the susceptibilitypolymorphisms in another graph. Thus, the SNP score can be negative ifthere are only susceptibility polymorphisms, positive, if there are onlyprotective polymorphisms, or either positive or negative, depending uponthe relative numbers of protective to susceptibility polymorphisms.

TABLE 10 Combined presence or absence of protective and susceptibilitypolymorphisms Score combining protective and susceptibilitypolymorphisms −2 −1 0 1 2 3 OCOPD n = 124 8 28 33 39 11 5 Resistant n =101 2 11 23 27 23 15 % OCOPD 80% 72% 59% 59% 32% 25%

II. Example 3 Case Association Study—Lung Cancer Methods SubjectRecruitment

Subjects of European decent who had smoked a minimum of fifteen packyears and diagnosed with lung cancer were recruited. Subjects met thefollowing criteria: diagnosed with lung cancer based on radiological andhistological grounds, including primary lung cancers with histologicaltypes of small cell lung cancer, squamous cell lung cancer,adenocarinoma of the lung, non-small cell cancer (where histologicalmarkers can not distinguish the subtype) and broncho-alveolar carcinoma.Subjects can be of any age and at any stage of treatment after thediagnosis had been confirmed. One hundred and nine subjects wererecruited, of these 58% were male, the mean FEV1/FVC (±95% confidencelimits) was 51% (49-53), mean FEV1 as a percentage of predicted was 43(41-45). Mean age, cigarettes per day and pack year history was 65 yrs(64-66), 24 cigarettes/day (22-25) and 50 pack years (41-55)respectively. Two hundred and seventeen European subjects who had smokeda minimum of twenty pack years and who had never suffered breathlessnessand had not been diagnosed with an obstructive lung disease or lungcancer in the past were also studied. This control group was recruitedthrough clubs for the elderly and consisted of 63% male, the meanFEV1/FVC (95% CI) was 82% (81-83), mean FEV1 as a percentage ofpredicted was 96 (95-97). Mean age, cigarettes per day and pack yearhistory was 59 yrs (57-61), 24 cigarettes/day (22-26) and 42 pack years(39-45) respectively. Using a PCR based method [1], all subjects weregenotyped for the α1-antitrypsin mutations (S and Z alleles) and thosewith the ZZ allele were excluded. 190 European blood donors (smokingstatus unknown) were recruited consecutively through local blood donorservices. Sixty-three percent were men and their mean age was 50 years.On regression analysis, the age difference and pack years differenceobserved between lung cancer sufferers and resistant smokers was foundnot to determine FEV or lung cancer.

This study shows that polymorphisms found in greater frequency in lungcancer patients compared to resistant smokers can reflect an increasedsusceptibility to the development of lung cancer. Similarly,polymorphisms found in greater frequency in resistant smokers comparedto lung cancer can reflect a protective role.

Summary of Characteristics.

Parameter Lung Cancer Resistant smokers Median (IQR) N = 109 N = 200Differences % male 52% 64% ns Age (yrs) 68 (11) 60 (12) P < 0.05 Packyears 40 (31) 43 (25) P < 0.05 Cigarettes/day 18 (11) 24 (12) ns FEV1(L) 1.7 (0.6) 2.8 (0.7) P < 0.05 FEV1 % predict 67 (22) 96% (10)    P <0.05 FEV1/FVC 59 (14) 82 (8)  P < 0.05

Means and 95% confidence limits

Glutathione S-Transferase Null Polymorphisms Genotyping

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described [7,herein incorporated by reference in its entirety]. Genotyping was doneusing minor modifications of the above protocol optimised for our ownlaboratory conditions The PCR reactions were amplified in MJ Researchthermocyclers in a total volume of 25 μl and contained 80 ng genomicDNA, 100 ng forward and reverse primers, 200 mM dNTPs, 20 mM Tris-HCL(pH 8.4), 50 mM KCl, 2.5 mM MgCl2 and 1.0 unit of Taq polymerase(Qiagen). Forward, internal (GSTM4) and reverse prime sequences were 5′CTGCCCTACTTGATTGATGG-3′ [SEQ.ID.NO.192], 5′ ATCTTCTCCTCTTCTGTCTC-3′[SEQ.ID.NO.193] and 5′-TTCTGGATTGTAGCAGATCA-3′ [SEQ.ID.NO.194]. Cyclingconditions consisted of 94 C 60 s, 59 C 30 s, 72 C 30 s for 35 cycleswith an extended last extension of 3 min. Digested products wereseparated on 3% agarose gel. The products were visualised by ultraviolettransillumination following ethidium bromide staining and migrationcompared against a 1 Kb plus ladder standard (Invitrogen). Genotypeswere recorded in data spreadsheets and statistical analysis performed.

Cyclooxygenase 2 Polymorphisms Genotyping

Genomic DNA was extracted from whole blood samples (Maniatis, T.,Fritsch, E. F. and Sambrook, J., Molecular Cloning Manual. 1989). TheCyclo-oxygenase 2 −765 polymorphism was determined by minormodifications of a previously published method (Papafili A, et al.,2002, incorporated in its entirety herein by reference)). The PCRreaction was carried out in a total volume of 25 ul and contained 20 nggenomic DNA, 500 pmol forward and reverse primers, 0.2 mM dNTPs, 10 mMTris-HCL (pH 8.4), 150 mM KCl, 1.0 mM MgCl₂ and 1 unit of polymerase(Life Technologies). Cycling times were incubations for 3 min at 95° C.followed by 33 cycles of 50 s at 94° C., 60 s at 66° C. and 60 s at 72°C. A final elongation of 10 min at 72° C. then followed. 4 ul of PCRproducts were visualised by ultraviolet trans-illumination of a 3%agarose gel stained with ethidium bromide. An aliquot of 3 ul ofamplification product was digested for 1 hr with 4 units of AciI (RocheDiagnostics, New Zealand) at 37° C. Digested products were separated ona 2.5% agarose gel run for 2.0 hours at 80 mV with TBE buffer. Theproducts were visualised against a 123 bp ladder using ultraviolettransillumination after ethidium bromide staining.

Matrix Metalloproteinase 1 −1607 1G/2G Polymorphisms Genotyping

Genomic DNA was extracted using standard phenol and chloroform methods.Cohorts of patients and controls were configured in to 96-well PCRformat containing strategic negative controls. The assay primers, PCRconditions and RFLP assays details have been previously described(Dunleavey, L. et al. Rapid genotype analysis of the matrixmetalloproteinase-1 gene 1G/2G polymorphism that is associated with riskof cancer. Matrix Biol. 19(2):175-7 (2000), herein incorporated byreference in its entirety). Genotyping was done using minormodifications of the above protocol optimised for our own laboratoryconditions The PCR reactions were amplified in MJ Research thermocyclersin a total volume of 25 μl and contained 80 ng genomic DNA, 100 ngforward and reverse primers, 200 mM dNTPs, 20 mM Tris-HCL (pH 8.4), 50mM KCl, 1.5 mM MgCl₂ and 1.0 unit of Taq polymerase (Qiagen). Forwardand reverse prime sequences were 3′ TCGTGAGAATGTCTTCCCATT-3′[SEQ.ID.NO.195] and 5′-TCTTGGATTGATTTGAGATAAGTGAAATC-3′[SEQ.ID.NO.196].Cycling conditions consisted of 94 C 60 s, 55 C 30 s, 72 C 30 s for 35cycles with an extended last extension of 3 min Aliquots ofamplification product were digested for 4 hrs with 6 Units of therestriction enzymes XmnI (Roche Diagnostics, New Zealand) at designatedtemperature conditions. Digested products were separated on 6%polyacrylamide gel. The products were visualised by ultraviolettransillumination following ethidium bromide staining and migrationcompared against a 1 Kb plus ladder standard (Invitrogen). Genotypeswere recorded in data spreadsheets and statistical analysis performed.

Polymorphism Genotyping Using the Sequenom Autoflex Mass Spectrometer

Genomic DNA was extracted from whole blood samples [2]. Purified genomicDNA was aliquoted (10 ng/ul concentration) into 96 well plates andgenotyped on a Sequenom™ system (Sequenom™ Autoflex Mass Spectrometerand Samsung 24 pin nanodispenser) using the following sequences,amplification conditions and methods. The following conditions were usedfor the PCR multiplex reaction: final concentrations were for 10× Buffer15 mM MgCl2 1.25×, 25 mM MgCl2 1.625 mM, dNTP mix 25 mM 500 uM, primers4 uM 100 nM, Taq polymerase (Quiagen hot start) 0.15 U/reaction, GenomicDNA 10 ng/ul. Cycling times were 95° C. for 15 min, (5° C. for 15 s, 56°C. 30 s, 72° C. 30 s for 45 cycles with a prolonged extension time of 3min to finish. We used shrimp alkaline phosphotase (SAP) treatment (2 ulto 5 ul per PCR reaction) incubated at 35° C. for 30 min and extensionreaction (add 2 ul to 7 ul after SAP treatment) with the followingvolumes per reaction of: water, 0.76 ul; hME 10× termination buffer, 0.2ul; hME primer (10 uM), 1 ul; MassEXTEND enzyme, 0.04 ul.

Sequenom Conditions for the Polymorphisms Genotyping—1

TERM SNP_ID 2nd-PCRP 1st-PCRP ACT CYP2E1_1019G/CPst1ACGTTGGATGAAACCAGAGGGAAGCAAAGG ACGTTGGATGTCATTGGTTGTGCTGCACCT [SEQ. ID.NO. 197] [SEQ. ID. NO. 198] ACT XPD-751 G/TACGTTGGATGCACCAGGAACCGTTTATGGC ACGTTGGATGAGCAGCTAGAATCAGAGGAG [SEQ. ID.NO. 199] [SEQ. ID. NO. 200] ACT IL-18 105 A/CACGTTGGATGGTCAATGAAGAGAACTTGGTC ACGTTGGATGAATGTTTATTGTAGAAAACC [SEQ. ID.NO. 201] [SEQ. ID. NO. 202] ACT IL-18-133 G/CACGTTGGATGGGGTATTCATAAGCTGAAAC ACGTTGGATGCCTTCAAGTTCAGTGGTCAG [SEQ. ID.NO. 203] [SEQ. ID. NO. 204] ACT CYP 1A1 Ile462ValACGTTGGATGGTGATTATCTTTGGCATGGG ACGTTGGATGGGATAGCCAGGAAGAGAAAG [SEQ. ID.NO. 205] [SEQ. ID. NO. 206] ACT MMP12 Asn 357 Ser A/GACGTTGGATGCCCTATTTCTTTGTCTTCAC ACGTTGGATGCTTGGGATAATTTGGCTCTG [SEQ. ID.NO. 207] [SEQ. ID. NO. 208] ACT OGG1 Ser 326 Cys G/CACGTTGGATGGGAACCCTTTCTGCGCTTTG ACGTTGGATGCCTACAGGTGCTGTTCAGTG [SEQ. ID.NO. 209] [SEQ. ID. NO. 210] ACT NAT2 Arg 197 Gln A/GACGTTGGATGCCTGCCAAAGAAGAAACACC ACGTTGGATGACGTCTGCAGGTATGTATTC [SEQ. ID.NO. 211] [SEQ. ID. NO. 212] ACT CYP2E1_C/T Rsa1ACGTTGGATGGTTCTTAATTCATAGGTTGC ACGTTGGATGCTTCATTTCTCATCATATTTTC [SEQ.ID. NO. 213] [SEQ. ID. NO. 214] ACG CCND1 A870GACGTTGGATGTAGGTGTCTCCCCCTGTAAG ACGTTGGATGTCCTCTCCAGAGTGATCAAG [SEQ. ID.NO. 215] [SEQ. ID. NO. 216] ACG ILB1-511 A/GACGTTGGATGATTTTCTCCTCAGAGGCTCC ACGTTGGATGTGTCTGTATTGAGGGTGTGG [SEQ. ID.NO. 217] [SEQ. ID. NO. 218] ACG FAS_A-670GACGTTGGATGTTGTGGCTGCAACATGAGAG ACGTTGGATGCTATGGCGCAACATCTGTAC [SEQ. ID.NO. 219] [SEQ. ID. NO. 220] ACG NOS3-786 T/CACGTTGGATGACTGTAGTTTCCCTAGTCCC ACGTTGGATGAGTCAGCAGAGAGACTAGGG [SEQ. ID.NO. 221] [SEQ. ID. NO. 222] ACT ACT_Ala15ThrACGTTGGATGGAGTTGAGAATGGAGAGAATG ACGTTGGATGTCAAGTGGGCTGTTAGGGTG [SEQ. ID.NO. 223] [SEQ. ID. NO. 224] ACT SOD3 Arg 312 GlnACGTTGGATGTGCTGCGTGGTGGGCGTGTG ACGTTGGATGGGCCTTGCACTCGCTCTCG [SEQ. ID.NO. 225] [SEQ. ID. NO. 226] ACT NOS3 Asp 298 GluACGTTGGATGAAACGGTCGCTTCGACGTGC ACGTTGGATGACCTCAAGGACCAGCTCGG [SEQ. ID.NO. 227] [SEQ. ID. NO. 228] CGT IL-8-251 A/TACGTTGGATGACTGAAGCTCCACAATTTGG ACGTTGGATGGCCACTCTAGTACTATATCTG [SEQ. ID.NO. 229] [SEQ. ID. NO. 230] CGT IFN gamma 874 A/TACGTTGGATGCAGACATTCACAATTGATTT ACGTTGGATGGATAGTTCCAAACATGTGCG [SEQ. ID.NO. 231] [SEQ. ID. NO. 232] ACT XRCC1 Arg 399 Gln G/AACGTTGGATGTAAGGAGTGGGTGCTGGACT ACGTTGGATGAGGATAAGGAGCAGGGTTGG [SEQ. ID.NO. 233] [SEQ. ID. NO. 234]

Sequenom Conditions for the Polymorphisms Genotyping—2

SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC PWARN UEP_DIR UEP_MASSCYP2E1_1019G/CPst 1 119 95.2 71.3 46.7 47.1 F 5256.4 XPD -751 G/T 11397.6 71.3 49.8 47.4 F 5689.7 IL-18 105 A/C 120 65.6 71.3 49.8 36.4 R6702.4 IL-18 -133 G/C 112 93.5 81.3 47.1 42.1 F 5811.8 CYP 1A1 Ile462Val102 98.2 81.3 55.6 55 F 6222.1 MMP12 Asn 357 Ser A/G 95 92.6 81.3 4830.4 F 7070.6 OGG1 Ser 326 Cys G/C 99 96.5 82.2 58.9 70.6 R 5227.4 NAT2Arg 197 Gln A/G 115 97.4 70 48.5 36.4 F 6635.3 CYP2E1_C/T Rsa1 105 62.877.8 46.4 26.1 R 7018.6 CCND1 A870G 106 98.1 83 45.8 47.1 R 5034.3 ILB1-511 A/G 111 99.2 83 46 47.1 R 5203.4 FAS_A-670G 103 99.2 83 54.4 50 R6166 NOS3 -786 T/C 114 97.5 83 61.8 61.9 F 6358.1 ACT_Ala15Thr 118 93.468.2 45.2 47.1 F 5136.4 SOD3 Arg 312 Gln 119 63.2 68.2 55.5 57.9 F5855.8 NOS3 Asp 298 Glu 113 82.2 68.2 65.4 66.7 F 6432.2 IL-8 -251 A/T119 92.6 75.8 45.9 28.6 R 6428.2 IFN gamma 874 A/T 112 75.3 75.8 46.426.1 F 6943.6 XRCC1 Arg 399 Gln G/A 109 93.6 93.6 66.8 82.4 F 5099.3

Sequenom Conditions for the Polymorphisms Genotyping—3

EXT1 EXT1 EXT2 SNP_ID UEP_SEQ CALL MASS EXT1_SEQ CALL CYP2E1_1019G/CPst1TTCTTGGTTCAGGAGAG C 5529.6 TTCTTGGTTCAGGAGAGC G [SEQ. ID. NO. 235] [SEQ.ID. NO. 236] XPD-751 G/T GCAATCTGCTCTATCCTCT T 5977.9GCAATCTGCTCTATCCTCTT G [SEQ. ID. NO. 237] [SEQ. ID. NO. 238] IL-18 105A/C ATTCAAGCTTGCCAAAGTAATC A 6990.6 ATTCAAGCTTGCCAAAGTAAT C [SEQ. ID.NO. 239] CT [SEQ. ID. NO. 240] IL-18 -133 G/C CATAAGCTGAAACTTCTGG C 6085CATAAGCTGAAACTTCTGGC G [SEQ. ID. NO. 241] [SEQ. ID. NO. 242] CYP 1A1Ile462Val GGAAGTGTATCGGTGAGACC A 6519.3 GGAAGTGTATCGGTGAGACC G [SEQ. ID.NO. 243] A [SEQ. ID. NO. 244] MMP12 Asn 357 Ser TGACAAATACTGGTTAATTAGCAA 7367.8 TGACAAATACTGGTTAATTAG G A/G [SEQ. ID. NO. 245] CAA [SEQ. ID.NO. 246] OGG1 Ser 326 Cys GCTCCTGAGCATGGCGG G 5500.6 GCTCCTGAGCATGGCGGCC G/C [SEQ. ID. NO. 247] [SEQ. ID. NO. 248] NAT2 Arg 197 GlnTACTTATTTACGCTTGAACCTC A 6932.5 TACTTATTTACGCTTGAACCT G A/G [SEQ. ID.NO. 249] CA [SEQ. ID. NO. 250] CYP2E1_C/T Rsa1 CTTAATTCATAGGTTGCAATTTT T7315.8 CTTAATTCATAGGTTGCAATT C [SEQ. ID. NO. 251] TTA [SEQ. ID. NO. 252]CCND1 A870G ACATCACCCTCACTTAC[SEQ. ID. G 5307.5 ACATCACCCTCACTTACC A NO.253] [SEQ. ID. NO. 254] ILB1 -511 A/G AATTGACAGAGAGCTCC G 5476.6AATTGACAGAGAGCTCCC A [SEQ. ID. NO. 255] [SEQ. ID. NO. 256] FAS_A-670GATGAGAGGCTCACAGACGTT G 6439.2 ATGAGAGGCTCACAGACGTT A [SEQ. ID. NO. 257]C [SEQ. ID. NO. 258] NOS3-786 T/C GGCATCAAGCTCTTCCCTGGC C 6631.3GGCATCAAGCTCTTCCCTGG T [SEQ. ID. NO. 259] CC [SEQ. ID. NO. 260]ACT_Ala15Thr GAATGTTACCTCTCCTG[SEQ. ID. A 5433.6 GAATGTTACCTCTCCTGA GNO. 261] [SEQ. ID. NO. 262] SOD3 Arg 312 Gln GCACTCAGAGCGCAAGAAG C 6129GCACTCAGAGCGCAAGAAGC G [SEQ. ID. NO. 263] [SEQ. ID. NO. 264] NOS3 Asp298 Glu GCTGCTGCAGGCCCCAGATGA T 6720.4 GCTGCTGCAGGCCCCAGATG G [SEQ. ID.NO. 265] AT [SEQ. ID. NO. 266] IL-8 -251 A/T CACAATTTGGTGAATTATCAA A6716.4 CACAATTTGGTGAATTATCAA T [SEQ. ID. NO. 267] T [SEQ. ID. NO. 268]IFN gamma 874 A/T TTCTTACAACACAAAATCAAATC T 7231.8 TTCTTACAACACAAAATCAAAA [SEQ. ID. NO. 269] TCT [SEQ. ID. NO. 270] XRCC1 Arg 399 GlnTCGGCGGCTGCCCTCCC A 5396.5 TCGGCGGCTGCCCTCCCA G G/A [SEQ. ID. NO. 271][SEQ. ID. NO. 272]

Sequenom Conditions for the Polymorphisms Genotyping—4

SNP_ID EXT2_MASS EXT2_SEQ 1stPAUSE CYP2E1_1019G/CPst1 5873.8TTCTTGGTTCAGGAGAGGT[SEQ. ID. NO. 273] 5585.6 XPD -751 G/T 6292.1GCAATCTGCTCTATCCTCTGC[SEQ. ID. NO. 274] 6018.9 IL-18 105 A/C 7658ATTCAAGCTTGCCAAAGTAATCGGA[SEQ. ID. 7031.6 NO. 275] IL-18 -133 G/C 6438.2CATAAGCTGAAACTTCTGGGA[SEQ. ID. NO. 6141 276] CYP 1A1 Ile462Val 6839.5GGAAGTGTATCGGTGAGACCGT[SEQ. ID. NO. 6551.3 277] MMP12 Asn 357 Ser A/G7688 TGACAAATACTGGTTAATTAGCAGT[SEQ. ID. 7399.8 NO. 278] OGG1 Ser 326 CysG/C 5853.8 GCTCCTGAGCATGGCGGGA[SEQ. ID. NO. 279] 5556.6 NAT2 Arg 197 GlnA/G 7261.8 TACTTATTTACGCTTGAACCTCGA[SEQ. ID. 6964.5 NO. 280] CYP2E1_C/TRsa1 7636 CTTAATTCATAGGTTGCAATTTTGT[SEQ. ID. 7347.8 NO. 281] CCND1 A870G5651.7 ACATCACCCTCACTTACTG[SEQ. ID. NO. 282] 5338.5 ILB1 -511 A/G 5820.8AATTGACAGAGAGCTCCTG[SEQ. ID. NO. 283] 5507.6 FAS_A-670G 6743.4ATGAGAGGCTCACAGACGTTTC[SEQ. ID. NO. 6470.2 284] NOS3-786 T/C 6975.5GGCATCAAGCTCTTCCCTGGCTG[SEQ. ID. 6662.3 NO. 285] ACT_Ala15Thr 5738.7GAATGTTACCTCTCCTGGC[SEQ. ID. NO. 286] 5465.6 SOD3 Arg 312 Gln 7116.6GCACTCAGAGCGCAAGAAGGGGC[SEQ. ID. 6185 NO. 287] NOS3 Asp 298 Glu 7034.6GCTGCTGCAGGCCCCAGATGAGC[SEQ. ID. 6761.4 NO. 288] IL-8 -251 A/T 7029.6CACAATTTGGTGAATTATCAAAT[SEQ. ID. NO. 6741.4 289] IFN gamma 874 A/T 7530TTCTTACAACACAAAATCAAATCAC[SEQ. ID. 7256.8 NO. 290] XRCC1 Arg 399 Gln G/A6054.9 TCGGCGGCTGCCCTCCCGGA[SEQ. ID. NO. 5428.5 291]

Sequenom Conditions for the Polymorphisms Genotyping—5

TERM SNP_ID 2nd-PCRP 1st-PCRP ACT CTGF-447G/C ACGTTGGATGAGGTAGCTGAAGAGACGTTGGATGGCCTATAGCCTCTAA GCAAAC [SEQ. ID. NO. 292] AACGC [SEQ. ID. NO.293] ACT NBS1 Gln185Glu ACGTTGGATGCTTTCAATTTGTGGAACGTTGGATGTGTGCACTCATTTGT G/C GGCTG [SEQ. ID. NO. 294] GGACG [SEQ. ID.NO. 295] ACT MBL2 161 G/A ACGTTGGATGGTAGCTCTCCAGGCAACGTTGGATGGTACCTGGTTCCCCC TCAAC [SEQ. ID. NO. 296] TTTTC [SEQ. ID. NO.297] ACT IGF2R Leu252Val ACGTTGGATGACACCAGGCGTTTGAACGTTGGATGAAAAACGCCAACAGC C/G TGTTG [SEQ. ID. NO. 298] ATCGG [SEQ. ID.NO. 299] ACT MUC5AC -221 C/T ACGTTGGATGAGGCGGAGATGGGTACGTTGGATGAGTCTAGGGTGGGG GTGTC [SEQ. ID. NO. 300] TATGTG [SEQ. ID. NO.301] ACT Arg1 intron1 C/T ACGTTGGATGATGTGTGGATTCACAACGTTGGATGGGGTTGGCAACTCTA GCTCG [SEQ. ID. NO. 302] AAAGG [SEQ. ID. NO.303] ACT REV1 Phe257Ser ACGTTGGATGCTCTGAAATCAGTGCACGTTGGATGATGGTCAACAGTGTT C/T TGCTC [SEQ. ID. NO. 304] GCCAG [SEQ. ID.NO. 305] ACT Apex1 Asp148Glu ACGTTGGATGCACCTCTTGATTGCTACGTTGGATGACCCGGCCTTCCTGA G/T TTCCC [SEQ. ID. NO. 306] TCATG [SEQ. ID.NO. 307] ACG IL-10 -1082 A/G ACGTTGGATGATTCCATGGAGGCTGACGTTGGATGGACAACACTACTAAG GATAG [SEQ. ID. NO. 308] GCTTC [SEQ. ID. NO.309]

Sequenom Conditions for the Polymorphisms Genotyping—6

SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC PWARN UEP_DIR UEP_MASSCTGF-447G/C 119 98.2 65 52 52.9 R 5090.3 NBS1 Gln185Glu G/C 118 97 65 5152.9 R 5192.4 MBL2 161 G/A 99 96.8 65 50.3 52.9 F 5299.5 IGF2R Leu252ValC/G 114 98.5 67.8 68.6 82.4 F 5206.4 MUC5AC -221 C/T 119 81.8 67.8 56.964.7 g R 5273.4 Arg1 intron1 C/T 102 99.6 67.8 53.3 52.6 R 5932.9 REV1Phe257Ser C/T 105 99.6 67.8 57.5 55 R 6003.9 Apex1 Asp148Glu G/T 11492.9 67.8 46.8 35 F 6113 IL-10 -1082 A/G 107 98 68.8 51.2 58.8 R 4977.2

Sequenom Conditions for the Polymorphisms Genotyping—7

SNP_ID UEP_SEQ EXT1_CALL EXT1_MASS EXT1_SEQ CTGF-447G/CAAAAGGTTTCTCCCCCC G 5363.5 AAAAGGTTTCTCCCCCCC [SEQ. ID. NO. 310] [SEQ.ID. NO. 311] NBS1 Gln185Glu AGGCTGCTTCTTGGACT G 5465.6AGGCTGCTTCTTGGACTC G/C [SEQ. ID. NO. 312] [SEQ. ID. NO. 313] MBL2 161G/A CAAAGATGGGCGTGATG A 5596.7 CAAAGATGGGCGTGATGA [SEQ. ID. NO. 314][SEQ. ID. NO. 315] IGF2R Leu252Val GCCAGCCCCGGGACGGA C 5479.6GCCAGCCCCGGGACGGA C/G [SEQ. ID. NO. 316] C [SEQ. ID. NO. 317] MUC5AC-221 ATGGGTGTGTCTGCCGG T 5570.6 ATGGGTGTGTCTGCCGGA C/T [SEQ. ID. NO.318] [SEQ. ID. NO. 319] Arg1 intron1 C/T GGCTGTAAGGAAATCTGGG T 6230.1GGCTGTAAGGAAATCTGG [SEQ. ID. NO. 320] GA [SEQ. ID. NO. 321] REV1Phe257Ser CCTTATCCTCCTCCTGGGAA T 6301.1 CCTTATCCTCCTCCTGGG C/T [SEQ. ID.NO. 322] AAA [SEQ. ID. NO. 323] Apex1 Asp148Glu TGTTTCATTTCTATAGGCGA T6401.2 TGTTTCATTTCTATAGGCG G/T [SEQ. ID. NO. 324] AT [SEQ. ID. NO. 325]IL-10 -1082 A/G CCTATCCCTACTTCCCC G 5250.4 CCTATCCCTACTTCCCCC [SEQ. ID.NO. 326] [SEQ. ID. NO. 327]Sequenom conditions for the polymorphisms Genotyping—8

SNP_ID EXT2_CALL EXT2_MASS EXT2_SEQ 1stPAUSE CTGF-447G/C C 5716.7AAAAGGTTTCTCCCCCCGA 5419.5 [SEQ. ID. NO. 328] NBS1 Gln185Glu C 5818.8AGGCTGCTTCTTGGACTGA 5521.6 G/C [SEQ. ID. NO. 329] MBL2 161 G/A G 5901.9CAAAGATGGGCGTGATGGC 5628.7 [SEQ. ID. NO. 330] IGF2R Leu252Val G 5823.8GCCAGCCCCGGGACGGAGT 5535.6 C/G [SEQ. ID. NO. 331] MUC5AC -221 C/T C5890.8 ATGGGTGTGTCTGCCGGGT 5602.6 [SEQ. ID. NO. 332] Arg1 intron1 C/T C6879.5 GGCTGTAAGGAAATCTGGGGGT 6262.1 [SEQ. ID. NO. 333] REV1 Phe257Ser C6630.3 CCTTATCCTCCTCCTGGGAAGA 6333.1 C/T [SEQ. ID. NO. 334] Apex1Asp148Glu G 7068.6 TGTTTCATTTCTATAGGCGAGGA 6442.2 G/T [SEQ. ID. NO. 335]IL-10 -1082 A/G A 5858.8 CCTATCCCTACTTCCCCTTC 5281.4 [SEQ. ID. NO. 336]

Results

Frequencies of individual polymorphisms are as follows:

TABLE 11 Polymorphism allele and genotype frequencies in the Lung cancerpatients, resistant smokers and controls. Nitric oxide synthase 3 Asp298 Glu (T/G) Allele* Genotype Frequency T G TT TG GG Controls n = 183(%) 108 (30%) 258 (70%) 13 (7%) 82 (45%) 88 (48%) Lung Cancer n = 107(%) 71 (33%) 143 (67%) 9 (8%) 53 (50%) 45 (42%) Resistant n = 198 (%)135 (34%) 261 (66%) 28^(1.2) (14%) 79 (40%) 91 (46%) Nitric oxidesynthase 3 −786 T/C Allele* Genotype Frequency C T CC CT TT Controls n =183 (%) Lung Cancer n = 107 (%) 82 (38%) 132 (62%) 16 (15%) 50 (47%) 41³(38%) Resistant n = 198 (%) 166 (42%) 228 (58%) 31 (16%) 104 (53%) 62(31%) Super oxide dismutase 3 Arg 312 Gln C/G Allele* Genotype FrequencyC G CC CG GG Controls n = 190 (%) 371 (98%) 9 (2%) 183 (96%) 5 (3%) 2(1%) Lung Cancer n = 104 (%) 208 (100%) 0 (0%) 104 (100%) 0 (0%) 0 (0%)Resistant n = 182 (%) 390 (98%) 10 (3%) 191 (95%) 8⁴ (4%) 1⁴ (1%) XRCC1Arg 399 Gln A/G Allele* Genotype Frequency A G AA AG GG Controls n = 190(%) Lung Cancer n = 103 (%) 68 (33%) 138 (67%) 4 (4%) 60 (58%) 39 (38%)Resistant n = 193 (%) 132 (34%) 254 (66%) 18⁵ (9%) 96 (50%) 79 (41%)Interleukin 8 −251 A/T Allele* Genotype Frequency A T AA AT TT Controlsn = 188 (%) 175 (47%) 201 (53%) 39 (21%) 97 (52%) 52 (28%) Lung Cancer n= 90 68 (38%) 112 (62%) 6 (7%) 56 (52%) 28 (31%) Resistant n = 199 (%)192⁷ (48%) 206 (52%) 45⁶ (23%) 102 (51%) 52 (26%) Anti-chymotrypsin Ala−15 Thr Allele* Genotype Frequency A G AA AG GG Lung Cancer n = 108 99(46%) 117⁹ (54%) 24 (22%) 51 (47%) 33⁸ (31%) Resistant n = 196 (%) 207(53%) 185 (47%) 52 (27%) 103 (53%) 41 (21%) Cyclin D1 A 870 G LungCancer n = 107 109 (51%) 105 (49%) 25¹¹ (23%) 59 (55%) 23 (21%)Resistant n = 199 (%) 188 (47%) 210 (53%) 45 (23%) 98 (49%) 56¹⁰ (28%)Interleukin 1B −511 A/G Lung Cancer n = 107 64 (30%) 150 (70%) 12 (11%)40 (37%) 55¹² (51%) Resistant n = 198 (%) 143 (36%) 253 (64%) 23 (12%)97 (49%) 78 (39%) FAS (Apo-1/CD 95) A −670 G Lung Cancer n = 106 121¹⁴(57%) 91 (43%) 32¹³ (30%) 57 (54%) 17 (16%) Resistant n = 198 (%) 202(51%) 194 (49%) 45 (23%) 112 (57%) 41 (21%) XPD 751 T/G Allele* GenotypeFrequency G T GG TG TT Lung Cancer n = 108 72 (33%) 144 (66%) 11 (10%)50 (46%) 47 (44%) Resistant n = 197 (%) 147 (37%) 247 (63%) 31¹⁵ (16%)85 (43%) 81 (41%) Cytochrome P450 1A1 Ile 462 Val G/A Allele* GenotypeFrequency G A GG AG AA Lung Cancer n = 109 5 (2%) 213 (98%) 0 (0%) 5(5%) 104¹⁶ (95%) Resistant n = 199 (%) 20 (5%) 378 (95%) 13¹⁶ (1%) 18¹⁶(9%) 180² (90%) MMP12 Asn 357 Ser Lung Cancer n = 109 8 (4%) 210 (96%) 1(1%) 6 (5%) 102 (94%) Resistant n = 199 (%) 21 (5%) 377 (95%) 0¹⁷ (0%)21¹⁷ (11%) 178 (89%) 8-oxoguanine DNA glycosylase Ser 326 Cys C/GAllele* Genotype Frequency G C GG CG CC Lung Cancer n = 109 40 (18%) 178(82%) 2 (2%) 36 (33%) 71 (65%) Resistant n = 199 (%) 100 (25%) 298 (75%)14¹⁸ (7%) 72 (36%) 113 (57%) N-Acetyltransferase 2 Arg 197 Gln G/AAllele* Genotype Frequency A G AA AG GG Lung Cancer n = 106 55 (26%) 157(74%) 9 (8%) 37 (35%) 60¹⁹ (57%) Resistant n = 195 (%) 122 (31%) 268(69%) 17 (9%) 88 (45%) 90 (46%) Cytochrome P450 2E1 1019 G/C Pst1Allele* Genotype Frequency C G CC CG GG Lung Cancer n = 109 10 (5%) 208(95%) 0 (0%) 10²⁰ (9%) 99 (91%) Resistant n = 197 (%) 11 (3%) 383 (97%)0 (0%) 11 (6%) 186 (94%) Cytochrome P450 2E1 C/T Rsa I Allele* GenotypeFrequency T C TT TC CC Lung Cancer n = 108 11 (5%) 205 (95%) 0 (0%) 11²¹(10%) 97 (90%) Resistant n = 198 (%) 11 (3%) 385 (97%) 0 (0%) 11 (6%)187 (94%) Interleukin 18 105 A/C Allele* Genotype Frequency C A CC AC AALung Cancer n = 107 50 (23%) 164 (77%) 8 (8%) 34 (33%) 65²² (61%)Resistant n = 200 (%) 116 (29%) 284 (71%) 17²² (9%) 82²² (41%) 101 (50%)Interleukin 18 −133 C/G Allele* Genotype Frequency G C GG CG CC LungCancer n = 109 52 (24%) 166 (76%) 8 (7%) 36 (33%) 65²³ (60%) Resistant n= 198 (%) 117 (30%) 279 (70%) 17²³ (9%) 83²³ (42%) 98 (49%) GlutathioneS-Transferase M null Allele* Frequency Null Wild Controls n = 178 75(42%) 103 (58%) Lung Cancer n = 107 67²⁴ (58%) 48 (42%) Resistant n =182 100 (55%) 82 (45%) Interferon-gamma 874 A/T Allele* GenotypeFrequency A T AA AT TT Controls n = 186 (%) 183 (49%) 189 (51%) 37 (20%)109 (58%) 40 (22%) Lung cancer n = 106 (%) 116 (55%) 96 (45%) 34^(25,26)(32%) 48 (45%) 24 (23%) Resistant n = 196 (%) 209 (53%) 183 (47%) 50(26%) 109 (56%) 37 (19%) Cyclooxygenase −765 C/G Allele* GenotypeFrequency C G CC CG GG Controls n = 95 (%) 27 (14%) 161 (86%) 3 (3%) 21(22%) 70 (75%) Lung Cancer n = 109 (%) 34 (16%) 184 (84%)³⁰ 5 (5%²⁷) 24(22%)²⁷ 80 (73%)²⁹ Resistant n = 158 (%) 75 (24%)²⁸ 241 (76%) 11 (7%) 53(34%) 94 (59%) Matrix metalloproteinase 1 −1607 1G/2G Allele* GenotypeFrequency 1G 2G 1G1G 1G2G 2G2G Controls n = 174 214 (61%) 134 (39%) 68(39%) 78 (45%) 28 (16%) (%) Lung Cancer n = 67 58 (43%) 76 (57%)³² 13(19%) 32 (48%) 22 (33%)³¹ (%) Resistant n = 171 167 (49%) 175 (51%) 41(24%) 85 (50%) 45 (26%) (%) *number of chromosomes (2n)

-   -   1. Genotype. TT vs TG/GG for resistant vs lung cancer, Odds        ratio (OR)=1.8, 95% confidence limits 0.8-4.3, χ² (Yates        uncorrected)=2.14, p=0.14, TT genotype=protective    -   2. Genotype. TT vs TG/GG for resistant vs controls, Odds ratio        (OR)=2.2, 95% confidence limits 1.0-4.6, χ² (Yates        corrected)=4.2, p=0.04, TT genotype=protective    -   3. Genotype. TT vs CC/CT for Lung cancer vs resistant, Odds        ratio (OR)=1.4, 95% confidence limits 0.8-2.3, χ² (Yates        uncorrected)=1.45, p=0.23, TT genotype=susceptible    -   4. Genotype CG/GG vs CC for resistant vs lung cancer, Yates        uncorrected=3.38, P=0.07 and Fisher's Two tailed test, P=0.03.        CG/GG=protective    -   5. Genotype. AA vs AG/GG for resistant vs lung cancer, Odds        ratio (OR)=2.6, 95% confidence limits 0.8-9.2, χ² (Yates        uncorrected)=2.89, p=0.09. AA genotype=protective    -   6. Genotype. AA vs AT/TT for resistant vs lung cancer, Odds        ratio (OR)=4.1, 95% confidence limits=1.6=11.2, χ² (Yates        corrected)=9.8, p=0.002, AA=protective    -   7. Allele. A vs T for resistant smokers vs lung cancer, Odds        ratio (OR)=1.5, 95% confidence limits 1.0-2.2, χ² (Yates        corrected)=5.07, p=0.02, A=protective    -   8. Genotype. GG vs AA/AG for Lung cancer vs resistant, Odds        ratio (OR)=1.7, 95% confidence limits=0.9-2.9, χ² (Yates        uncorrected)=3.51, p=0.06, GG=susceptible    -   9. Allele. G vs A for lung cancer vs resistant smokers, Odds        ratio (OR)=1.3, 95% confidence limits 0.9-1.9, χ² (Yates        uncorrected)=2.71, p=0.10,        -   G=susceptible    -   10. Genotype. GG vs AG/AA for Resistant vs lung cancer, Odds        ratio (OR)=1.4, 95% confidence limits=0.8-2.6, χ² (Yates        uncorrected)=1.6, p=0.20, GG=protective    -   11. Genotype. AG/AA vs GG for Lung cancer vs resistant, Odds        ratio (OR)=1.4, 95% confidence limits=0.8-2.6, χ² (Yates        uncorrected)=1.6, p=0.20, AA=susceptible    -   12. Genotype. GG vs AA/AG for Lung cancer vs resistant, Odds        ratio (OR)=1.6, 95% confidence limits=1-2.7, χ² (Yates        uncorrected)=4.07, p=0.04, GG=susceptible    -   13. Genotype. AA vs AG/GG for Lung cancer vs resistant, Odds        ratio (OR)=1.5, 95% confidence limits=0.8-2.6, χ² (Yates        uncorrected)=2.03, p=0.15, AA=susceptible    -   14. Allele. A vs G for Lung cancer vs resistant, Odds ratio        (OR)=1.3, 95% confidence limits 0.9-1.8, χ² (Yates        uncorrected)=2.04, p=0.15, A=susceptible    -   15. Genotype. GG vs TG/TT for Resistant vs lung cancer, Odds        ratio (OR)=1.7, 95% confidence limits=0.8-3.7, χ² (Yates        uncorrected)=1.81, p=0.18, GG=protective    -   16. Genotype. AG/GG vs AA for Resistant vs lung cancer, Odds        ratio (OR)=2.2, 95% confidence limits=0.7-6.9, χ² (Yates        uncorrected)=2.41, p=0.12, GG/AG=protective, AA=susceptible    -   17. Genotype. GG/AG vs AA for Resistant vs COPD, Odds ratio        (OR)=1.7, 95% confidence limits=0.7-4.6, χ² (Yates        uncorrected)=1.45, p=0.23, GG/AG=protective    -   18. Genotype. GG vs CG/CC for Resistant vs lung cancer, Odds        ratio (OR)=4.0, 95% confidence limits=0.9-26.3, χ² (Yates        uncorrected)=3.87, p=0.05, GG=protective    -   19. Genotype. GG vs AG/AA for Lung cancer vs resistant, Odds        ratio (OR)=1.5, 95% confidence limits=0.9-2.5, χ² (Yates        uncorrected)=3.0, p=0.08, GG=susceptible    -   20. Genotype. CG vs GG for Lung cancer and resistant, Odds ratio        (OR)=1.7, 95% confidence limits=0.7-4.5, χ² (Yates        uncorrected)=1.42, p=0.23, CG=susceptible    -   21. Genotype. TC vs CC for Lung cancer and resistant, Odds ratio        (OR)=1.9, 95% confidence limits=0.8-5.0, χ² (Yates        uncorrected)=2.24, p=0.13, TC=susceptible    -   22. Genotype. AA vs AC/CC for Lung cancer and resistant, Odds        ratio (OR)=1.6, 95% confidence limits=1.0-2.6, χ² (Yates        uncorrected)=3.51, p=0.06, AA=susceptible, AC/CC protective    -   23. Genotype. CC vs CG/GG for Lung cancer and resistant, Odds        ratio (OR)=1.5, 95% confidence limits=0.9-2.5, χ² (Yates        uncorrected)=2.90, p=0.09, CC=susceptible, CG/GG protective    -   24. Genotype. Null vs wild for Lung cancer and controls, Odds        ratio (OR)=1.92, 95% confidence limits=1.2-3.2, χ² (Yates        corrected)=6.64, p=0.01, Null=susceptible    -   25. Genotype. AA vs AT/TT for lung cancer vs resistant, Odds        ratio (OR)=1.4, 95% confidence limits 0.8-2.4, χ² (Yates        uncorrected)=1.48, p=0.22, AA genotype=susceptible    -   26. Genotype. AA vs AT/TT for lung cancer vs controls, Odds        ratio (OR)=1.9, 95% confidence limits 1.1-3.4, χ² (Yates        corrected)=5.45, p=0.02, AA genotype=susceptible to lung cancer    -   27. Genotype. CC/CG vs GG for Lung cancer vs resistant, Odds        ratio (OR)=0.53, 95% confidence limits=0.3-0.9, χ² (Yates        corrected)=4.9, P=0.03 CC/CG=protective    -   28. Allele. C vs G for Lung cancer vs resistant, Odds ratio        (OR)=0.59, 95% confidence limits 0.4-0.9, χ² (Yates        corrected)=4.8, p=0.03, C=protective    -   29. Genotype. GG vs CG/CC for Lung cancer vs resistant, Odds        ratio (OR)=1.88, 95% confidence limits=1.1-3.3, χ² (Yates        corrected)=4.9, P=0.03 GG=susceptible (when compared against        resistant smokers but not controls)    -   30. Allele. G vs C for Lung cancer vs resistant, Odds ratio        (OR)=1.7, 95% confidence limits 1.1-2.7, χ² (Yates        corrected)=4.8, p=0.03, G=susceptible (when compared against        resistant smokers but not controls)    -   31. Genotype. 2G2G vs 1G1G/1G2G for Lung cancer vs controls,        Odds ratio (OR)=2.55, 95% confidence limits 1.3-5.1, χ² (Yates        corrected)=7.3, p=0.007 2G2G genotype=susceptible    -   32. Allele. 2G vs 1G for Lung cancer vs controls, Odds ratio        (OR)=2.1, 95% confidence limits 1.4-3.2, χ² (Yates        corrected)=12.3, p=0.0004, 2G=susceptible

Connective Tissue Growth Factor (CTGF) −447 G/C Polymorphism Allele andGenotype Frequencies in the Lung Cancer and Resistant Smokers.

37. Allele* 38. Genotype Frequency G C GG GC CC Lung cancer n = 109 20117  92 17 0 (%) (92%) (8%) (84%) (16%) (0%) Resistant n = 200 379 21 17921 0 (%) (95%) (5%) (90%) (10%) (0%) *number of chromosomes (2n)

-   -   1. Genotype. GC/CC vs GG for lung cancer vs resistant, Odds        ratio (OR)=1.6, 95% confidence limits 0.8-3.3, χ² (Yates        uncorrected)=1.70, p=0.19, GC/CC genotype=susceptibility (trend)

Mucin 5AC (Muc5AC)-221 C/T Polymorphism Allele and Genotype Frequenciesin the Lung Cancer and Resistant Smokers.

39. Allele* 40. Genotype Frequency C T CC CT TT Lung cancer n = 109 17741  73 31  5 (%) (81%) (19%) (67%) (28%) (5%) Resistant n = 195 296 94119 58 18 (%) (76%) (24%) (61%) (30%) (9%) *number of chromosomes (2n)

-   -   1. Genotype. TT vs CC/CT for lung cancer vs resistant, Odds        ratio (OR)=0.47, 95% confidence limits 0.2-1.4, χ² (Yates        uncorrected)=2.16, p=0.14, TT genotype=protective (trend)

Mannose Binding Lectin (MBL2) 161 G/A Polymorphism Allele and GenotypeFrequencies in the Lung Cancer and Resistant Smokers.

41. Allele* 42. Genotype Frequency G A GG AG AA Lung cancer n = 105 17337  71 31 3 (%) (82%) (18%) (67%) (30%) (3%) Resistant n = 197 338 56147 44 6 (%) (86%) (14%) (75%) (22%) (3%) *number of chromosomes (2n)

-   -   1. Genotype. AG/AA vs GG for lung cancer vs resistant, Odds        ratio (OR)=1.4, 95% confidence limits 0.8-2.4, χ² (Yates        uncorrected)=1.67, p=0.20, AG/AA genotype=susceptibility (trend)

Nibrin (NBS1) Gln185Glu G/C Polymorphism Allele and Genotype Frequenciesin the Lung Cancer and Resistant Smokers.

43. Allele* 44. Genotype Frequency G C GG GC CC Lung cancer n = 109 150 68  54 42 13 (%) (69%) (31%) (50%) (39%) (12%) Resistant n = 199 295103 107 81 11 (%) (74%) (26%) (54%) (41%)  (6%) *number of chromosomes(2n)

-   -   1. Genotype. CC vs CG/GG for lung cancer vs resistant, Odds        ratio (OR)=2.3, 95% confidence limits 0.9-5.8, χ² (Yates        uncorrected)=4.01, p=0.05, CC genotype=susceptibility

Arginase 1 (Arg1) Intron 1 C/T Polymorphism Allele and GenotypeFrequencies in the Lung Cancer and Resistant Smokers.

45. Allele* 46. Genotype Frequency C T CC CT TT Lung cancer n = 105 137 73 45 47 13 (%) (65%) (35%) (43%) (45%) (12%) Resistant n = 180 203 15765 73 42 (%) (56%) (44%) (36%) (41%) (23%) *number of chromosomes (2n)

-   -   1. Genotype. TT vs CC/CT for lung cancer vs resistant, Odds        ratio (OR)=0.46, 95% confidence limits 0.2-0.95, χ² (Yates        uncorrected)=5.11, p=0.02, TT genotype=protective    -   2. Allele. T vs C for lung cancer vs resistant, Odds ratio        (OR)=0.69, 95% confidence limits 0.5-1.0, χ² (Yates        corrected)=3.96, p=0.05, T allele=protective

REV1 Phe257Ser C/T Polymorphism Allele and Genotype Frequencies in theLung Cancer and Resistant Smokers.

47. Allele* 48. Genotype Frequency C T CC CT TT Lung cancer n = 109 12989 39 51 19 (%) (59%) (41%) (36%) (47%) (17%) Resistant n = 192 242 14283 76 33 (%) (63%) (37%) (43%) (40%) (17%) *number of chromosomes (2n)

-   -   1. Genotype. CC vs CT/TT for lung cancer vs resistant, Odds        ratio (OR)=0.73, 95% confidence limits 0.4-1.2, χ² (Yates        uncorrected)=1.6, p=0.20, CC genotype=protective (trend)

Insulin-Like Growth Factor II Receptor (IGF2R) Leu252Val C/GPolymorphism Allele and Genotype Frequencies in the Lung Cancer andResistant Smokers.

49. Allele* 50. Genotype Frequency C G CC CG GG Lung cancer n = 109 19028  82 26 1 (%) (87%) (13%) (75%) (24%) (1%) Resistant n = 198 342 54150 42 6 (%) (86%) (14%) (76%) (21%) (3%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs CC/CG for lung cancer vs resistant, Odds        ratio (OR)=0.30, 95% confidence limits 0.01-2.5, χ² (Yates        uncorrected)=1.41, p=0.22 (1-tailed t-test), GG        genotype=protective (trend)

Apex Nuclease (APE1) Asp148Glu T/G Polymorphism Allele and GenotypeFrequencies in the Lung Cancer and Resistant Smokers.

51. Allele* 52. Genotype Frequency T G TT TG GG Lung cancer n = 109 124 94 39 46 24 (%) (57%) (43%) (36%) (42%) (22%) Resistant n = 192 229 15569 91 32 (%) (60%) (40%) (36%) (47%) (17%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs TT/TG for lung cancer vs resistant, Odds        ratio (OR)=1.4, 95% confidence limits 0.8-2.7, χ² (Yates        uncorrected)=1.3, p=0.25, GG genotype=susceptibility (trend)        Interleukin 10 (IL-10)-1082 A/G polymorphism allele and genotype        frequencies in the lung cancer and resistant smokers.

53. Allele* 54. Genotype Frequency G C GG GC CC Lung cancer n = 98 91105 16 59 23 (%) (46%) (54%) (16%) (60%) (24%) Resistant n = 196 174 21840 94 62 (%) (44%) (56%) (20%) (48%) (32%) *number of chromosomes (2n)

-   -   1. Genotype. GG vs GC/CC for lung cancer vs resistant, Odds        ratio (OR)=0.66, 95% confidence limits 0.4-1.2, χ² (Yates        uncorrected)=2.12, p=0.15, GG genotype=protective (trend)

Table 12 below provides a summary of the protective and susceptibilitypolymorphisms determined for lung cancer.

TABLE 12 Summary of protective and susceptibility polymorphisms in LungCancer patients relative to resistant smokers (with normal lungfunction) Gene Polymorphism Role Nitric Oxide synthase 3 (NOS3) NOS3 Asp298 Glu TT protective Nitric Oxide synthase 3 (NOS3) NOS3 −786 T/C TTsusceptible Superoxide dismutase 3 (SOD3) SOD3 Arg 312 Gln CG/GGprotective XRCC1 XRCC1 Arg 399 Gln G/A AA protective Interleukin-8(IL-8) IL-8 −251 A/T AA protective Anti-chymotrypsin (ACT) ACT Ala 15Thr GG susceptible Cyclin D (CCND1) CCND1 A870G GG protective AAsusceptible Interleukin 1B (IL-1B) IL-1B −511 A/G GG susceptible FAS(Apo-1/CD95) FAS A-670G AA susceptible XPD XPD −751 G/T GG protectiveCYP 1A1 CYP 1A1 Ile 462 Val GG/AG protective A/G AA susceptible Matrixmetalloproteinase 12 MMP12 Asn 357 Ser GG/AG protective (MMP12) A/G8-Oxoguanine DNA glycolase OGG1 Ser 326 Cys G/C GG protective (OGG1)N-acetyltransferase 2 (NAT2) NAT2 Arg 197 Gln A/G GG susceptible CYP2E1CYP2E1 1019 G/C Pst I CC/CG susceptible CYP2E1 CYP2E1 C/T Rsa I TT/TCsusceptible Interleukin-18 (IL-18) IL-18 105 A/C AC/CC protective AAsusceptible Interleukin-18 (IL-18) IL-18 −133 G/C CG/GG protective CCsusceptible Glutathione S-transferase M GSTM null Null susceptibleInterferon gamma (IFN?) IFN? 874 A/T AA susceptible Cyclo-oxygenase 2(COX2) COX2 −765 G/C CC/CG protective GG susceptible Matrixmetalloproteinase 1 (MMP1) MMP −1607 1G/2G 2G2G susceptible Connectivetissue growth factor CTGF −447 G/C GC/CC (CTGF) susceptible Mucin 5AC(MUC5AC) MUC5AC −221 C/T TT protective Mannose binding lectin 2 (MBL2)MBL2 +161 G/A AG/AA susceptible Nibrin (NBS1) NBS1 Gln185Glu G/C CCsusceptible Arginase 1 (Arg1) Arg1 intron 1 C/T TT protective REV1 REV1Phe257Ser C/T CC protective Insulin-like growth factor II receptor IGF2RLeu252Val C/G GG protective (IGF2R) Apex nuclease (Apex or APE1)) ApexAsp148Glu G/T GG susceptible Interleukin 10 (IL-10) IL-10 −1082 A/G GGprotective

The combined frequencies of the presence or absence of the selectedprotective genotypes CYP1A1 GG/AG, OGG1 GG, CCND1 GG, NOS3 298 TT, IL-8AA, and XRCC1 AA observed in the subjects with lung cancer and inresistant smokers is presented below in Table 13.

TABLE 13 Combined frequencies of the presence or absence of selectedprotective genotypes in subjects with lung cancer and in resistantsmokers. Number of protective polymorphisms Cohorts 0 1 =2 Total LungCancer 66 (61%) 37 (34%) 6 (6%) 109 Resistant smokers 71 (36%) 86 (43%)42 (21%) 199 % of smokers with Lung 66/137 37/123 6/42 cancer (48%)(30%) (14%) Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+,Resist vs Lung — — 22.3 <0.0001 cancer 2+ vs 0-1, Resist vs Lung cancer4.6 1.8-12.5 11.87 0.0005 0 vs 2+, Lung cancer vs Resist 2.8 1.7-4.6 16.7 <0.0001

The combined frequencies of the presence or absence of the selectedsusceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-1B GG, and ACT 15GG, observed in the subjects with lung cancer and in resistant smokersis presented below in Table 14.

TABLE 14 Combined frequencies of the presence or absence of selectedsusceptibility genotypes in subjects with lung cancer and in resistantsmokers. Number of susceptibility polymorphisms Cohorts 0 1 =2 TotalLung Cancer 21 (19%) 52 (48%) 35 (33%) 108 Resistant smokers 71 (36%) 85(43%) 42 (21%) 198 % of smokers with 21/92 52/137 35/77 COPD (23%) (38%)(45%) Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, — — 10.20.006 Lung cancer vs Resist 2+ vs 0-1, Lung cancer vs Resist 1.8 1.0-3.14.1 0.04 0+ vs 1-2+, Resist vs COPD 2.3 1.3-4.2 8.2 0.004

The combined frequencies of the presence or absence of the selectedprotective genotypes CYP1A1 GG/AG, OGG1 GG, CCND1 GG, NOS3 298 TT, SOD3CG/GG, XPD GG, MMP12 GG/AG, and XRCC1 AA observed in the subjects withlung cancer and in resistant smokers is presented below in Table 15.

TABLE 15 Combined frequencies of the presence or absence of selectedprotective genotypes in subjects with lung cancer and in resistantsmokers. Number of protective polymorphisms n = 8 Cohorts 0 1 =2 TotalLung Cancer 54 (50%) 50 (46%) 5 (4%) 109 Resistant smokers 67 (34%) 83(42%) 50 (25%) 199 % of smokers with Lung 54/121 50/133 5/55 cancer(45%) (38%) (9%) Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+,Resist vs Lung — — 21.5 <0.0001 cancer 2+ vs 0-1, Resist vs Lung cancer6.9 2.5-20.5 18.7 <0.0001 0 vs 2+, Lung cancer vs Resist 2.0 1.2-3.2 6.96 0.008

The combined frequencies of the presence or absence of the selectedsusceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-1B GG, ACT 15 GG,NAT2 GG, IL-18 105 AA, and IFN? AA, observed in the subjects with lungcancer and in resistant smokers is presented below in Table 16.

TABLE 16 Combined frequencies of the presence or absence of selectedsusceptibility genotypes in subjects with lung cancer and in resistantsmokers. Number of susceptibility polymorphisms n = 7 Cohorts 1 2 =3Total Lung Cancer 16 (15%) 35 (32%) 58 (53%) 109 Resistant smokers 65(33%) 66 (33%) 69 (34%) 200 % of smokers with 16/81 35/101 58/127 COPD(20%) (35%) (46%) Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+,Lung cancer — — 14.6 0.0007 vs Resist 3+ vs 1-2, Lung cancer vs Resist2.2 1.3-5.6 9.4 0.002 1 vs 2-3+, Resist vs COPD 2.8 1.5-5.4 10.7 0.001

The combined frequencies of the presence or absence of the selectedprotective genotypes CYP1A1 GG/AG, OGG1 GG, CCND1 GG, NOS3 298 TT, IL-8AA, XRCC1 AA, and Cox 2 −765 CC/CG, observed in the subjects with lungcancer and in resistant smokers is presented below in Table 17.

TABLE 17 Combined frequencies of the presence or absence of protectivegenotypes in the exposed smoking subjects (Lung cancer subjects andresistant smokers). Number of protective genotypes Cohorts 0 1 =2 TotalLung Cancer 45 (40%) 50 (43%) 19 (17%) 114 Resistant smokers 47 (23%) 79(40%) 74 (37%) 200 % of smokers with Lung 45/92 50/129 19/93 cancer(49%) (39%) (20%) Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+,Resist vs Lung — — 16.8 0.0002 cancer 2+ vs 0-1, Resist vs Lung cancer2.94 1.6-5.4 13.44 0.0002 0 vs 2+, Lung cancer vs Resist 2.12 1.3-3.68.2 0.004

The combined frequencies of the presence or absence of the selectedsusceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-B1 GG, ACT 15 GG,and MMP1 2G2G, observed in the subjects with lung cancer and inresistant smokers is presented below in Table 18.

TABLE 18 Combined frequencies of the presence or absence ofsusceptibility genotypes in the exposed smoking subjects (Lung cancersubjects and resistant smokers). Number of susceptibility genotypesCohorts 0-1 2-3 4-6 Total Lung Cancer 13 (12%) 66 (61%) 30 (28%) 109Resistant smokers 54 (27%) 113 (56%) 33 (17%) 200 % of smokers with13/67 66/179 30/63 COPD (19%) (37%) (48%) Comparison Odd's ratio 95% CI?2 P value 0-1 vs 2-3 vs 4-6, — — 11.8 0.003 Lung cancer vs Resist 4-6vs rest, Lung cancer vs 1.9 1.0-3.5 4.6 0.03 Resist 0-1 vs rest, Resistvs COPD 2.7 1.4-5.6 8.6 0.003

Protective polymorphisms were assigned a score of −1 whilesusceptibility polymorphisms were assigned a score of +1. For eachsubject, a net score was then calculated according to the presence ofsusceptibility and protective genotypes. This produced a linear spreadof values, as shown in Table 14. When assessed as a range between −2 to+4, a linear relationship as depicted in FIG. 4 was observed. Thisanalysis indicates that for subjects with a net score of −2 or less,there was a minimal risk of having lung cancer. For subjects with a netscore of −1, there was an approximately one in ten risk of having lungcancer. In contrast, for subjects with a net score of 4+ or greater, therisk was markedly increased to over 70% (see Table 19 and FIG. 4). It isnoted that for FIG. 4, unlike the data presented in FIG. 3, theprotective polymorphisms are assigned a negative value while thesusceptibility polymorphisms are assigned a positive value. The precisevalue or sign given to each one is not critical, as long as it isconsistent between the types of polymorphisms.

TABLE 19 Combined presence or absence of protective and susceptibilitypolymorphisms Score combining protective (−1) and susceptibility (+1)polymorphisms −2 −1 0 1 2 3 4+ Lung cancer 0 (0%) 2 (2%) 10 (9%)  21(19%) 38 (35%) 23 (21%) 15 (14%) N = 109 (%) Resistant smokers 6 (3%) 21(11%) 39 (20%) 51 (26%) 52 (26%) 25 (13%) 6 (3%) N = 200 (%) % Lungcancer 0% 9% 20% 29% 42% 48% 71%

A further combined analysis was performed using a greater number ofpolymorphisms. Again, this produced a linear spread of values (Table20). When assessed as a range between −3 to +5, a linear relationship asdepicted in FIG. 5 was observed. This analysis indicates that forsubjects with a net score of −2 or less, there was a minimal risk ofhaving lung cancer. In contrast, for subjects with a net score of 5+ orgreater, the risk was markedly increased to 80% (see Table 20 and FIG.5).

TABLE 20 Combined presence or absence of protective and susceptibilitypolymorphisms SNP score for Lung cancer according to the presence ofprotective(−1) and susceptibility (+1) genotypes for all smokers Cohorts<−3 −2 −1 0 1 2 3 4 5+ Lung cancer 0 (0%)  1 (1%)  3 (3%) 10 (9%)  25(23%) 32 (29%) 20 (18%) 14 (13%) 4 (4%)   N = 109 Resistant smokers 3(2%) 12 (6%) 16 (8%) 34 (17%) 58 29%)  48 (24%) 21 (11%) 7 (4%) 1 (0.5%)N = 200 % Lung cancer 0% 7% 16% 23% 30% 40% 49% 67% 80%

DISCUSSION

The methods of the invention allow the determination of risk of diseaseto be assessed. For example, a simple scoring system in which eachpolymorphism in a category (i.e. protective or susceptibility) isassigned the same value allows the combined effects of all potentiallyrelevant polymorphisms to be factored into the analysis. In otherembodiments, the methods of the invention utilize a scoring system withadjustment (weighting) for the magnitude of the effect of eachindividual polymorphism, and again allow all polymorphisms to besimultaneously analyzed.

In other embodiments, analyses can utilise path analysis and/orMonte-Carlo analysis where the non-genetic and genetic factors can beanalyzed.

Similar results were observed in comparing the presence or absence ofsusceptibility and resistant polymorphisms in smokers with OCOPD, and insmokers with lung cancer and resistant smokers.

The benefit of a net susceptibility score, having been determined for asubject is that it provides the opportunity for early prophylacticand/or therapeutic intervention. Such intervention can be as simple ascommunicating the net susceptibility score to the subject together withan explanation of the implications of that score. This alone can cause alifestyle or occupational change, with the resultant beneficial effectfor the subject.

Other, more direct approaches to prophylaxis or therapy can also befollowed. These can include pharmaceutical or other medicaments beingadministered directed at favourably altering the net score of thesubject together with other such approaches as discussed herein.

Table 21 below presents representative examples of polymorphisms inlinkage disequilibrium with the polymorphisms specified herein. Examplesof such polymorphisms can be located using public databases, such asthat available online at world wide web dot hapmap dot org. Specifiedpolymorphisms are indicated in the columns marked SNP NAME. Uniqueidentifiers are indicated in the columns marked RS NUMBER.

TABLE 21 Polymorphisms reported to be in linkage disequilibrium (unlessstated) with examples of specified polymorphism. SNP NAME RS NUMBER SNPNAME RS NUMBER SNP NAME RS NUMBER COX2 SNPs rs6684912 rs5277 rs7527769rs2745559 rs2066823 rs7550380 rs12042763 rs4648263 rs2206594 rs4648250rs4987012 rs6687495 rs4648251 rs20428 rs6681231 rs2223626 rs20429rs13376484 rs689462 rs4648264 rs12064238 rs4648253 rs4648265 rs10911911rs689465 rs4648266 rs12743673 rs12027712 rs4648267 rs10911910 rs689466rs11567824 rs12743516 rs2745558 rs4648268 rs10911909 rs3918304 rs4648269rs1119066 rs20415 rs4648270 rs1119065 rs20416 rs12759220 rs1119064rs4648254 rs20430 rs10798053 rs11567815 rs4648271 rs12409744 −765G > Crs20417 rs11567825 rs10911908 rs4648256 rs4648273 rs10911907 rs20419rs16825748 rs7416022 rs2734779 rs4648274 rs2745561 rs20420 rs16825745rs10911906 rs20422 rs20432 rs2734776 rs20423 rs20433 rs2734777 rs5270rs3218622 rs12084433 rs20424 rs2066826 rs2734778 rs5271 rs5278 rs2745560rs4648257 rs4648276 rs2223627 rs11567819 rs20434 rs2383517 rs3134591rs3218623 rs4295848 rs3134592 rs3218624 rs4428839 rs20426 rs5279rs4609389 rs4648258 rs4648278 rs4428838 rs11567820 rs13306034 rs12131210rs2745557 rs2853803 rs2179555 rs11567821 rs4648279 rs2143417 rs4648259rs4648281 rs2143416 rs4648260 rs4648282 rs11583191 rs4648261 rs11567826rs2383516 rs4648262 rs4648283 rs2383515 rs11567822 rs4648284 rs10911905rs11567823 rs4648285 rs10911904 rs2066824 rs11567827 rs20427 rs4648286rs4648287 rs1042719 rs5744244 rs5272 rs3729944 rs360722 rs4648288rs3730182 rs5023207 rs5273 rs1042720 rs5744246 rs5274 rs6879202rs5744247 rs3218625 rs3777124 −133 C/G rs360721 rs4648289 rs1803051rs4988359 rs4648290 rs8192451 rs12721559 rs1051896 rs4987255 rs5744248rs5275 rs3177007 rs5744249 1ADRB SNPs rs1126871 rs5744250 rs2082382rs6885272 rs5744251 rs2082394 rs6889528 rs100000356 rs2082395 rs4521458rs1834481 rs9325119 rs10463409 rs17215057 rs9325120 rs7702861 rs5744253rs12189018 IL-18 SNPs rs5744254 rs11168066 rs187238 rs5744255 rs11959615rs5744228 rs5744256 rs11958940 rs360718 rs5744257 rs4705270 rs360717rs360720 rs10079142 rs5744229 rs5744258 rs9325121 rs100000353 rs5744259rs11746634 rs5744231 rs5744260 rs11168067 rs5744232 rs5744261 rs9325122rs7106524 105 A/C rs549908 rs11957351 rs189667 PAI-1 SNPs rs11948371rs12290658 rs6465787 rs11960649 rs12271175 rs7788533 rs1432622rs11606049 rs6975620 rs1432623 rs360716 rs6956010 rs11168068 rs360715rs12534508 rs17778257 rs360714 rs4729664 rs2400706 rs2043055 rs2527316rs2895795 rs5744233 rs2854235 rs2400707 rs795467 rs10228765 rs2053044rs12270240 rs2854225 rs17108803 rs100000354 rs2854226 rs12654778rs4937113 rs2227707 rs11168070 rs100000355 rs2227631 rs11959427 rs360723−675 4G/5G No rs rs1042711 rs5744237 NOS3 SNPs rs1801704 rs5744238rs2373962 Arg16Gly rs1042713 rs5744239 rs2373961 rs1042714 rs7932965rs6951150 rs1042717 rs11214103 rs13238512 rs1800888 rs5744241 rs10247107rs1042718 rs5744242 rs10276930 rs3729943 rs5744243 rs10277237 rs12703107rs9282804 rs2282679 rs6946340 Asp298Glu rs1799983 rs2282680 rs6946091VDBP SNPs rs705117 rs6946415 rs222035 rs2070741 rs10952296 rs222036rs2070742 rs13309715 rs16846943 rs6821541 rs10952297 rs7668653 rs222048rs7784943 rs1491720 rs432031 rs11771443 rs16845007 rs432035 rs2243310rs17830803 rs222049 rs1800783 Glu416Asp rs7041 rs222050 rs3918155Lys420Thr rs4588 rs12510584 rs3918156 rs3737553 rs17467825 rs2566519rs9016 GSTP1 SNPs rs3918157 rs1352846 rs656652 rs3918158 rs222039rs625978 rs3918159 rs3775154 rs6591251 rs2566516 rs222040 rs12278098rs3918225 rs843005 rs612020 rs3918160 rs222041 rs12284337 rs1800779rs7672977 rs12574108 rs2243311 rs705121 rs6591252 rs3918161 rs11723621rs597717 rs10952298 rs2298850 rs688489 rs2070744 rs705120 rs597297rs3918226 rs2298851 rs6591253 rs3918162 rs844806 rs6591254 rs3918163rs1491709 rs7927381 rs3918164 rs705119 rs7940813 rs3918165 rs6845925rs593055 rs1800781 rs12640255 rs7927657 rs13310854 rs12644050 rs614080rs13310763 rs6845869 rs7941395 rs2853797 rs12640179 rs7941648 rs13311166rs222042 rs7945035 rs13310774 rs3187319 rs2370141 rs2853798 rs222043rs2370142 rs11974098 rs842999 rs7949394 rs3918166 rs222044 rs7949587rs3730001 rs222045 rs6591255 rs3918167 rs16846912 rs8191430 rs3918168rs222046 rs6591256 rs3918169 rs705118 rs8191431 rs3918170 rs222047rs8191432 rs3793342 rs13142062 rs7109914 rs3793341 rs843000 rs4147580rs1549758 rs3755967 rs8191436 rs1007311 rs1491710 rs8191437 rs9282803rs2282678 rs17593068 rs8191438 rs2069718 rs7145047 rs8191439 rs3087272rs7141735 rs8191440 rs2069719 rs11558264 rs8191441 rs9282708 rs6647rs1079719 rs2069720 rs8350 rs1871041 rs1042274 rs2230075 rs4147581rs2069721 rs1049800 rs8191444 rs2069734 S allele rs17580 rs8191445rs2069722 rs2854258 rs2370143 rs2234687 rs2753937 rs8191446 rs7957366rs2749547 rs3891249 rs2069723 rs1243162 rs8191447 rs2069724 rs2753938rs12796085 rs2069725 rs2070709 rs8191448 rs4394909 rs17090719 rs762803rs2069726 rs11846959 rs8191449 rs2069727 rs1802962 Ile105Val rs947894IL-13 SNPs rs2749521 rs4986948 −1055 C/T rs1800925 rs2753939 rs675554rs11575055 rs1802959 rs749174 rs2069755 rs1802961 rs8191450 rs2069741rs1050469 rs743679 rs2069742 Z allele no rs rs1799811 rs2069743rs1050520 rs11553890 rs2069756 rs12077 rs4986949 rs3212142 rs12233rs8191451 rs2066960 rs13170 rs1871042 rs1295687 rs1303 rs11553892rs3212145 rs1802960 rs4891 rs2069744 rs1243163 rs6413486 rs2069745rs2073333 rs5031031 rs2069746 rs1243164 rs947895 rs2069747 rs7144409IFN-SNPs rs2069748 rs7142803 rs2069707 rs1295686 rs1243165 rs3814242Arg130Gln rs20541 rs1051052 rs2069709 rs2069749 rs1243166 rs2069710rs1295685 rs11628917 rs2069711 rs848 rs11832 rs2069712 rs2069750rs9944155 874 A/T rs2430561 rs847 1237 G/A rs11568814 rs2069713a1-antitrypsin rs877081 SNPs rs1861494 rs709932 rs877082 rs2234685rs11558261 rs877083 rs1861493 rs20546 rs877084 rs2069714 rs11558263rs875989 rs2069715 F1028580 rs9944117 rs2069716 rs7145770 rs1884546rs2069717 rs2239652 rs1884547 rs1885065 rs2735442 rs8046608 rs1884548rs2569693 rs5743264 rs1243167 rs281439 rs5743266 rs17751614 rs281440rs2076752 rs1884549 rs2569694 rs5743267 rs1243168 rs11575073 rs8061316rs17090693 rs2569695 rs8061636 rs17824597 rs2075741 rs16948754 TNFa SNPsrs11575074 rs7206340 rs1799964 rs2569696 rs2076753 rs1800630 rs2735439rs2067085 rs1799724 rs2569697 rs16948755 +489 G/A rs1800610 rs2075742rs2111235 rs3093662 rs2569698 rs2111234 rs3093664 rs11669397 rs7190413−308 G/A rs1800629 (1) rs901886 rs7206582 SMAD3 SNPs rs885742 rs8045009C89Y C89Y no rs (2) rs2569699 rs6500328 ICAM1 rs1056538 rs7500036rs1799969 rs11549918 rs8057341 rs5493 rs2569700 rs12918060 rs5030381rs2228615 rs7204911 rs5494 rs2569701 rs7500826 rs3093033 rs2569702rs4785449 rs5495 rs2735440 rs12922299 rs1801714 rs2569703 rs11649521rs13306429 rs10418913 rs13339578 rs2071441 rs1056536 rs17221417 rs5496rs2569704 rs13331327 rs5497 rs11673661 rs11642482 rs13306430 rs2569705rs11642646 E469K rs5498 rs10402760 rs17312836 rs5030400 rs2569706rs5743268 rs2071440 rs2569707 rs5743269 rs5499 rs2735441 rs5743270rs3093032 rs2436545 rs12925051 rs1057981 rs2436546 rs12929565 rs5500rs2916060 rs13380733 rs5501 rs2916059 rs13380741 rs5030383 rs2916058rs11647841 rs281436 rs2569708 rs10451131 rs923366 rs12972990 rs2066842rs281437 rs735747 rs5743271 rs3093030 rs885743 rs7498256 rs5030384 NOD2SNPs rs5743272 rs5030385 rs4785224 rs5743273 rs3810159 rs5743261rs2076754 rs281438 rs5743262 rs2066843 rs3093029 rs5743263 rs1078327rs5743274 rs11645386 rs1031101 rs1861759 rs7187857 rs10824795 rs5743275rs8061960 rs10824794 rs5743276 rs5743294 rs920725 rs2066844 rs2357791rs7916582 rs5743277 rs7359452 rs920724 rs5743278 rs7203344 rs16933335rs6413461 rs5743295 rs11003125 rs3813758 rs5743296 rs7100749 rs5743279rs3135499 rs11003124 rs5743280 rs5743297 rs7084554 rs5743281 rs5743298rs7096206 rs4785225 rs5743299 rs11003123 rs16948773 rs3135500 rs11575988rs9931711 rs5743300 rs11575989 rs17313265 rs8056611 rs7095891 rs11646168rs2357792 rs4647963 rs9925315 rs12600253 rs8179079 rs5743284 rs12598306rs5030737 rs5743285 rs7205423 161 G/A rs1800450 rs751271 rs718226rs1800451 rs748855 MBL2 SNPs rs12246310 rs1861758 rs7899547 rs12255312rs13332952 rs10824797 rs11003122 rs7198979 rs11003131 rs1982267rs1861757 rs930506 rs1982266 rs7203691 rs930505 rs4935047 rs5743286rs11003130 rs4935046 rs5743287 rs2384044 rs10824793 rs10521209 rs2384045rs1838066 Gly881Arg rs2066845 rs5027257 rs1838065 rs5743289 rs2384046rs930509 rs8063130 rs12263867 rs930508 rs2076756 rs11003129 rs930507rs12920425 rs12221393 CMA1 SNPs rs12920040 rs2165811 rs1956920rs12920558 rs12782244 rs1956921 rs12919099 rs11003128 −1903 G/Ars1800875 rs12920721 rs17664818 rs1800876 rs2076755 rs7475766 rs3759635rs5743290 rs10824796 rs1956922 rs5743291 rs16933417 rs1956923 rs11642651rs2165810 NAT2 SNPs rs1861756 rs11003127 rs11780272 rs749910 rs3925313rs2101857 rs4990643 rs7094151 rs13363820 rs1077861 rs7071882 rs6984200rs5743292 rs12264958 rs13277605 rs9921146 rs11003126 rs9987109 rs7820330rs7596849 −366 G/A rs9550373 rs7460995 rs4848306 rs11542984 rs2087852rs3087257 rs4769055 rs2101684 rs7556811 rs17074937 rs7011792 rs7556903rs9671065 rs1390358 rs6743438 rs9579645 rs923796 rs6743427 rs9579646rs4546703 rs6761336 rs4075131 rs4634684 rs6761335 rs4075132 rs2410556rs6743338 rs9315043 rs11996129 rs6761245 rs9315044 rs4621844 rs6761237rs4597169 rs11785247 rs6743330 rs9578037 rs1115783 rs6743326 rs9578196rs1115784 rs6743322 rs4293222 rs1961456 rs6761220 rs10507391 rs1112005rs6761218 rs12429692 rs11782802 rs5021469 rs4769871 rs973874 rs6710598rs4769872 rs1495744 rs1143623 rs4769873 rs7832071 rs1143624 rs12430051rs1805158 rs2708920 rs9315045 rs1801279 rs1143625 rs9670278 rs1041983rs2853545 rs4503649 rs1801280 rs2708921 rs9508832 rs4986996 rs1143626rs9670460 rs12720065 rs3087258 rs3885907 rs4986997 C-511T rs16944rs3922435 rs1799929 rs3917346 rs9551957 Arg197Gln rs1799930 rs4986962rs12018461 rs1208 rs1143627 rs9551958 rs1799931 MEH SNPs rs10467440rs2552 Tyr113His rs1051740 (2) rs12017304 rs4646247 His139Arg rs2234922(2) rs9551959 rs971473 ALOX5AP SNPs rs11617473 rs721398 rs4076128rs11147438 IL-1B SNPs rs9508830 rs10162089 rs10169916 rs4073259rs9551960 rs13009179 rs4073260 rs9285075 rs4849127 rs11616333 rs12431114rs4849126 rs4073261 rs4254165 rs7558108 rs4075474 rs4360791 rs13032029rs4075473 rs17612031 rs13013349 rs9670115 rs3803277 rs12623093 rs9315042rs3803278 rs3087255 rs3809376 rs12429469 rs3087256 rs12877064 rs17612099rs6721954 rs9508831 rs9550576 rs12621220 rs9670503 rs4356336 rs4584668rs2075800 rs2734714 rs4238137 CLCA1 SNPs rs6661730 rs17612127 rs2791519rs2753377 rs4147063 rs2791518 rs2753378 rs4147064 rs5744302 rs2145412rs4147062 rs1321697 rs2180762 rs9315046 rs2753338 rs1005569 rs9506352rs2791517 rs5744325 rs9670531 rs5744303 rs5744326 rs9671182 rs2734706rs1985554 rs9315047 rs2753345 rs1985555 rs17690694 rs2753347 rs100000102rs9652070 rs2753348 rs100000103 rs17074966 rs2753349 rs1969719 rs4387455rs5744304 rs2390102 rs4254166 rs5744305 rs5744329 rs4075692 rs1358826rs1407142 rs17690748 rs2753359 rs2753384 rs9671124 rs5744306 rs2753385rs9671125 rs2734711 rs5744330 rs9741436 rs5744307 rs5744331 rs9578197rs2734712 rs926064 rs4769056 rs2753361 rs926065 rs11147439 rs2753364rs926066 rs12721459 rs1555389 rs926067 rs4769874 rs2753365 rs2753386HSP70 HOM SNPs rs100000100 rs2180764 rs1043618 rs100000101 rs2734689rs11576009 rs5744310 rs5744332 rs11557922 rs5744311 rs5744333 rs11576010rs5744312 rs11161837 rs1008438 rs4656114 rs5744335 rs11576011 rs5744313rs2038485 rs4713489 rs2753367 rs3765989 rs16867582 rs4656115 rs2734690rs12526722 rs2734713 rs5744336 rs6933097 rs5744314 rs2734691 rs12213612rs5744315 rs2734692 rs481825 rs5744316 rs5744337 rs7757853 rs5744317rs5744338 rs7757496 rs5744318 rs2734694 rs9469057 rs926063 rs5744339rs12182397 rs5744319 rs100000104 rs16867580 rs5744320 rs2791515rs2075799 rs5744321 rs4656116 rs482145 rs5744322 rs5744342 rs2227957rs5744323 rs5744343 T2437C rs2227956 rs5744324 rs2180761 rs2227955rs2791516 rs5744344 rs5744345 rs5744443 rs6032038 rs1358825 rs5744444rs6032039 rs2145410 rs3138074 rs2267863 rs2734695 rs13166911 rs6124692rs5744346 rs2563310 +49 C/T No rs rs5744347 rs2569193 rs17333103rs100000105 rs2569192 rs17333180 rs5744349 rs5744446 rs1983649 rs4655913rs5744447 rs16989785 rs1321696 rs5744448 rs17424356 rs5744352 rs3138076rs6017500 rs11583355 rs12519656 rs6032040 rs100000106 rs5744449rs6017501 rs1321695 rs2915863 rs2664581 +13924 T/A rs1321694 rs3138078rs17424474 rs2791514 rs6875483 rs17333381 rs2734696 rs2569191 rs1053826rs5744354 rs5744451 rs2664533 rs2791513 rs5744452 rs1053831 rs2753332rs100000098 rs2664520 rs2791512 rs17118968 rs2267864 rs2791511 rs5744455rs13038355 rs2734697 −159 C/T rs2569190 rs13043296 CD14 SNPs rs2569189rs13039213 rs6877461 rs2563303 rs6104049 rs3822356 rs3138079 rs13043503rs6877437 rs2228049 rs6104050 rs12153256 rs13763 rs17424578 rs11554680rs11556179 rs17424613 rs12109040 rs4914 rs6017502 rs12517200 Elafin SNPsrs6094101 rs5744430 rs2868237 rs6130778 rs5744431 rs4632412 rs6130779rs100000092 rs7347427 rs6104051 rs5744433 rs6032032 rs6104052rs100000093 rs10854230 ADBR2 SNPs rs4912717 rs7347426 rs2082382rs100000094 rs8183548 rs2082394 rs100000095 rs6104047 rs2082395rs100000096 rs6513967 rs9325119 rs6864930 rs13038813 rs9325120rs100000097 rs8118673 rs12189018 rs6864583 rs7346463 rs11168066rs6864580 rs7362841 rs11959615 rs6889418 rs13042694 rs11958940 rs6889416rs13038342 rs4705270 rs5744440 rs7363327 rs10079142 rs5744441 rs6073668rs9325121 rs5744442 rs13044826 rs11746634 rs11168067 rs1800468 rs542603rs9325122 rs4987025 rs574939 rs11957351 rs1800469 rs573764 rs11948371rs11466314 rs7102189 rs11960649 rs12977628 rs575727 rs1432622 rs12977601rs552306 rs1432623 rs12985978 rs634607 rs11168068 rs11466315 rs12286876rs17778257 rs11551223 rs12285331 rs2400706 rs11551226 rs519806 rs2895795rs11466316 rs12283571 rs2400707 rs13306706 rs2839969 rs2053044rs13306707 rs2000609 rs17108803 rs13306708 rs7125865 rs12654778rs9282871 rs570662 rs11168070 Leu10Pro rs1982073 rs11225427 rs11959427rs1800471 rs484915 rs1042711 rs13447341 rs470307 rs1801704 rs11466318rs2408490 rs1042713 rs12976890 rs12279710 Gln27Glu rs1042714 rs12978333rs685265 rs1042717 rs10420084 rs7107224 rs1800888 rs10418010 rs1155764rs1042718 rs12983775 rs534191 SOD3 SNPs rs12462166 rs509332 Arg213Glyrs1799895 (2) rs2241715 rs12283759 TGFB1 SNPs rs9749548 rs2105581rs1529717 rs7258445 rs470206 rs1046909 rs11466320 rs533621 rs2241712rs11466321 −1607 G/GG rs1799750 rs2241713 rs8108052 rs470211 rs2241714rs6508976 rs470146 rs11673525 rs8108632 rs2075847 rs2873369 rs11466324rs473509 rs11083617 rs2241716 rs498186 rs11083616 rs2241717 GSTM1polymorphism rs4803458 rs2288873 Null Null allele No rs (2) rs11670143rs12973435 MMP9 SNPs rs1982072 rs2014015 rs11696804 rs11668109 rs1989457rs6104416 rs13345981 rs10406816 rs3933239 rs11666933 rs8102918 rs3933240rs11466310 rs4803455 rs6094237 rs11466311 MMP1 SNPs rs11697325 rs2317130rs529381 rs6130988 rs4803457 rs1144396 rs6073983 rs3087453 rs504875rs6130989 rs1800820 rs526215 rs6130990 rs1054797 rs12280880 rs10211842rs6073984 rs8125587 TIMP3 SNPs rs6073985 rs3918253 rs5754289 rs8121146rs2274755 rs5754290 rs6032620 rs2664538 rs9606994 rs11698788 rs3918254rs7285034 rs6032621 rs6130993 rs13433582 rs6065912 rs3918255 rs1962223rs6104417 rs2236416 rs8137129 rs3848720 rs6130994 rs1807471 rs13040272rs3918256 rs7290885 rs6104418 rs3918281 rs5749511 rs3848721 rs3787268rs11703366 rs3848722 rs3918257 rs4990774 rs6104419 rs6017725 −1296 T/Crs9619311 rs4810482 rs6032623 rs2234921 rs3761157 rs3918258 rs2234920rs3761158 rs2250889 rs16991235 rs3761159 rs3918259 rs4638893 rs8113877rs3918260 rs12169569 rs6065913 rs13969 rs5998639 rs6104420 rs6104427rs7284166 rs6104421 rs6104428 rs5749512 rs3918240 rs2274756 rs6104422rs6017726 rs3918278 rs3918261 rs3918241 rs6032624 −1562 C/T rs3918242rs3918262 rs3918243 rs3918263 rs3918279 rs3918264 rs3918280 rs6130995rs4578914 rs6130996 rs6017724 rs3918265 rs3918244 rs3918266 rs3918245rs3918267 rs6130992 rs6073987 rs3918247 rs6073988 rs3918248 rs3918282rs3918249 rs1802909 rs6104423 rs13925 rs6104424 rs20544 rs6104425rs1056628 rs6104426 rs1802908 rs3918250 rs2664517 rs1805089 rs9509rs3918251 rs3918268 rs13040572 rs3918269 rs13040580 rs3918270 rs3918252MMP12 SNPs rs8125581 −82 A/G rs2276109 (2) (1 = no other SNPs reportedto be in LD, 2 = no other SNPS reported to be in LD)

INDUSTRIAL APPLICATION

The present invention is directed to methods for assessing a subject'srisk of developing a disease. The methods include the analysis ofpolymorphisms herein shown to be associated with increased or decreasedrisk of developing a disease, or the analysis of results obtained fromsuch an analysis, and the determination of a net risk score. Methods oftreating subjects at risk of developing a disease herein described arealso provided. Additional information regarding the above material, orsubparts thereof, can be found in U.S. patent application Ser. No.10/479,525, filed Jun. 16, 2004; and PCT Application No. PCT/NZ02/00106,filed Jun. 5, 2002, which further designates New Zealand Application No.512169, filed Jun. 5, 2001; New Zealand Application No. 513016, filedJul. 17, 2001, and New Zealand Application No. 514275, filed Sep. 18,2001, all of which are incorporated by reference in their entireties.Additional information can also be found in PCT application Nos. ______and ______, filed May 10, 2006, entitled “Methods and Compositions forAssessment of Pulmonary Function and Disorders” and “Methods of Analysisof Polymorphisms and Uses Thereof” respectively, having Agent ReferenceNos. 542813JBM and 542814JBM respectively, both of which areincorporated in their entireties by reference. PCT Application AgentReference No. 542813JBM claims priority to: NZ application No. 539934,filed May 10, 2005; NZ application No. 541935, filed Aug. 19, 2005; andJP application No. 2005-360523, filed Dec. 14, 2005, all of which areincorporated by reference in their entireties. PCT Application AgentReference No. 542814JBM claims priority to: NZ application No. 540249,filed May 20, 2005; and NZ application No. 541842, filed Aug. 15, 2005,all of which are incorporated in their entireties by reference.Additional information can also be found in U.S. Pat. No. ______, filedconcurrently with the instant application, entitled “Methods andCompositions for Assessment of Pulmonary Function and Disorders”,attorney docket No; SGENZ.013AUS, incorporated by reference in itsentirety.

PUBLICATIONS

-   1. Sandford A J, et al., 1999. Z and S mutations of the    α1-antitrypsin gene and the risk of chronic obstructive pulmonary    disease. Am J Respir Cell Mol. Biol. 20; 287-291.-   2. Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning    Manual. 1989.-   3. Papafili A, et al., 2002. Common promoter variant in    cyclooxygenase-2 represses gene expression. Arterioscler Thromb Vasc    Biol. 20; 1631-1635.-   4. Ukkola, O., Erkkilä, P. H., Savolainen, M. J. &    Kesäniemi, Y. A. 2001. Lack of association between polymorphisms of    catalase, copper zinc superoxide dismutase (SOD), extracellular SOD    and endothelial nitric oxide synthase genes and macroangiopathy in    patients with type 2 diabetes mellitus. J Int Med 249; 451-459.-   5. Smith C A D & Harrison D J, 1997. Association between    polymorphism in gene for microsomal epoxide hydrolase and    susceptibility to emphysema. Lancet. 350; 630-633.-   6. Lorenz E, et al., 2001. Determination of the TLR4 genotype using    allele-specific PRC. Biotechniques. 31; 22-24.-   7. Cantlay A M, Smith C A, Wallace W A, Yap P L, Lamb D, Harrison    D J. Heterogeneous expression and polymorphic genotype of    glutathione S-transferases in human lung. Thorax. 1994,    49(10):1010-4.

All patents, publications, scientific articles, and other documents andmaterials referenced or mentioned herein are indicative of the levels ofskill of those skilled in the art to which the invention pertains, andeach such referenced document and material is hereby incorporated byreference to the same extent as if it had been incorporated by referencein its entirety individually or set forth herein in its entirety. Anyand all materials and information from any such patents, publications,scientific articles, web sites, electronically available information,and other referenced materials or documents can be physicallyincorporated into this specification.

The specific methods and compositions described herein arerepresentative of various embodiments or preferred embodiments and areexemplary only and not intended as limitations on the scope of theinvention. Other objects, aspects, examples and embodiments will occurto those skilled in the art upon consideration of this specification,and are encompassed within the spirit of the invention as defined by thescope of the claims. It will be readily apparent to one skilled in theart that varying substitutions and modifications can be made to theinvention disclosed herein without departing from the scope and spiritof the invention. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, orlimitation or limitations, which is not specifically disclosed herein asessential. Thus, for example, in each instance herein, in embodiments orexamples of the present invention, any of the terms “comprising”,“consisting essentially of”, and “consisting of” can be replaced witheither of the other two terms in the specification, thus indicatingadditional examples, having different scope, of various alternativeembodiments of the invention. Also, the terms “comprising”, “including”,containing”, etc. are to be read expansively and without limitation. Themethods and processes illustratively described herein suitably can bepracticed in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality (for example, a culture or population)of such host cells, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by theApplicant.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedcan be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended indicative claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following indicative claims. Inaddition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

1. A method of assessing a subject's risk of developing a disease whichcomprises: analysing a biological sample from said subject for thepresence or absence of a protective polymorphism and for the presence orabsence of a susceptibility polymorphism, wherein said protectivepolymorphism and said susceptibility polymorphism are associated withsaid disease; assigning a positive score for each protectivepolymorphism and a negative score for each susceptibility polymorphismor vice versa; calculating a net score for said subject, said net scorerepresenting the balance between the combined value of the protectivepolymorphism and the combined value of the susceptibility polymorphismpresent in the subject sample; wherein a net protective score ispredictive of a reduced risk of developing said disease and a netsusceptibility score is predictive of an increased risk of developingsaid disease.
 2. A method according to claim 1 wherein the valueassigned to each protective polymorphism is the same. 3.-25. (canceled)26. A method of determining a subject's risk of developing a disease,said method comprising: obtaining the result of one or more analyses ofa sample from said subject to determine a presence or absence of aprotective polymorphism and a presence or absence of a susceptibilitypolymorphism, and wherein said protective and susceptibilitypolymorphisms are associated with said disease; assigning a positivescore for each protective polymorphism and a negative score for eachsusceptibility polymorphism or vice versa; and calculating a net scorefor said subject, said net score representing a balance between acombined value of the protective polymorphism and a combined value ofthe susceptibility polymorphism present in the subject sample, wherein anet protective score is predictive of a reduced risk of developing saiddisease and a net susceptibility score is predictive of an increasedrisk of developing said disease.
 27. (canceled)
 28. A method oftreatment of a subject to decrease a risk of developing a diseasethrough alteration of the net score for said subject as determined by amethod as defined above, wherein said method of treatment comprises:reversing, genotypically or phenotypically, the presence and/orfunctional effect of one or more susceptibility polymorphisms associatedwith said disease; and/or replicating and/or mimicking, genotypically orphenotypically, the presence and/or functional effect of one or moreprotective polymorphisms associated with said disease 3) a) reversing,genotypically or phenotypically, the presence, functional effect, orpresence and functional effect of one or more susceptibilitypolymorphisms associated with said disease and b) replicating,mimicking, or replicating and mimicking, genotypically orphenotypically, the presence, functional effect, or presence andfunctional effect of one or more protective polymorphisms associatedwith said disease.
 29. (canceled)